Progress
Cloning | Transfection | Induction | Cytometry | Data Analysis |
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| | | | |
Background
The BCR setup that we used in iGEM (CD79X-TCS-Gal4VP16 and Syk-15-TEVp) resulted in an inverted signal i.e. lower output when the BCR was activated with antiIgM than when unactivated. We expect that this inverted signal was a result of how fusing proteins to TEVp and TCS affected the sterics of their interaction. We think that when Syk was recruited to the activated BCR, it was no longer able to access the TCS, resulting in lower signal than background. This experiment aims to optimize the relative positions of the system's components by finding the optimal fusion protein linker lengths. We want to find the optimal length of the CD79-XnTCSXc-Gal4VP16 linker (where Xn and Xc are the number of amino acids of the n terminal and c terminal side of the TCS, respectively) and the Syk-TEVp linker. Optimizing 3 factors simultaneously gives a 3D search space as shown below. We chose data points in a way that maximized the space explored in the least number of points.
Image Added
Image Added
Approach
For ease of screening to identify correct linker constructs, we want to clone Protein1-pTet:mRFP-Protein2 constructs.
Parts
Part | h:CA-pT:RFP-G4 | h:CA-pT:RFP-G4 | TRE:Syk-pT:RFP-G4 |
Status | | Need SDM to correct BsaI site in Gal4 | |
Part | hEF1a:Heavy | hEF1a:Light | UAS:mKate |
Status | | | |
hEF1a:CD79A-XnTCSXc-Gal4VP16
3TCS3 | | 9TCS3 | | 15TCS3 |
| | | | |
| 6TCS6 | 9TCS6 | 12TCS6 | |
| | | | |
3TCS9 | 6TCS9 | 9TCS9 | 12TCS9 | 15TCS9 |
| | | | |
| 6TCS12 | 9TCS12 | 12TCS12 | |
| | | | |
3TCS15 | | 9TCS15 | | 15TCS15 |
| | | | |
TRE:Syk-X-tTEVp
Procedure
Controls
Untransfected | Single Color Blue | Single Color Red | Double Color | -VE control (background activation) | -VE control (background cleavage) | +VE control |
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HEK293 | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:eBFP hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk-15-tTEVp
Dox x uM | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:mKate hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk-15-tTEVp
Dox x uM | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn |
...
hEF1a:eBFP hEF1a:mKate hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk-15-tTEVp
Dox x uM | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate hEF1a:CD79A
hEF1a:CD79B TRE:Syk-15-tTEVp
Dox x uM | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk
Dox x uM | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate hEF1a:CD79A hEF1a:CD79B TRE:Syk-15-tTEVp hEF1a:GAl4VP16
Dox x uM |
* Maximum linker lengths were used in controls to evaluate the maximum background activation (CD79A-15TCS15-Gal4VP16) and maximum background cleavage (Syk-15-tTEVp)
Experimental Conditions
- anti IgM | + anti IgM |
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HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate hEF1a:CD79A-XnTCSXc-Gal4VP16 hEF1a:CD79B TRE:Syk-Xs-tTEVp dox x uM | HEK293 hEF1a:rtTA hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate hEF1a:CD79A-XnTCSXc-Gal4VP16 hEF1a:CD79B TRE:Syk-Xs-tTEVp
dox x uM anti IgM x uM |
Xs | XnTCSXc |
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S3 | 3tcs3, 3tcs15, 9tcs9, 15tcs3, 15tcs15 |
S6 | 6tcs6, 6tcs12, 9tcs9, 12tcs6, 12tcs12 |
S9 | 3tcs9, 6tcs9, 9tcs3, 9tcs6, 9tcs9, 9tcs12, 9tcs15, 12tcs9, 15tcs9 |
S12 | 6tcs6, 6tcs12, 9tcs9, 12tcs6, 12tcs12 |
S15 | 3tcs3, 3tcs15, 9tcs9, 15tcs3, 15tcs15 |
Expand |
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Plate 1 Plate 2 WELL 8 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3tcs3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 9 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3tcs3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA50ng
Dox 1 nM anti IgM x uM | WELL 10 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3tcs15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 11 HEK293 hEF1a:rtTA |
|
...
Results
Discussion
Background
The BCR setup that we used in iGEM (CD79X-TCS-Gal4VP16 and Syk-15-TEVp) resulted in an inverted signal i.e. lower output when the BCR was activated with antiIgM than when unactivated. We expect that this inverted signal was a result of how fusing proteins to TEVp and TCS affected the sterics of their interaction. We think that when Syk was recruited to the activated BCR, it was no longer able to access the TCS, resulting in lower signal than background. This experiment aims to optimize the relative positions of the system's components by finding the optimal fusion protein linker lengths. We want to find the optimal length of the CD79-XnTCSXc-Gal4VP16 linker (where Xn and Xc are the number of amino acids of the n terminal and c terminal side of the TCS, respectively) and the Syk-TEVp linker. Optimizing 3 factors simultaneously gives a 3D search space as shown below. We chose data points in a way that maximized the space explored in the least number of points.
Image Removed
Approach
For ease of screening to identify correct linker constructs, we want to clone Protein1-pTet:mRFP-Protein2 constructs.
Parts
Part | h:CA-pT:RFP-G4 | h:CA-pT:RFP-G4 | TRE:Syk-pT:RFP-G4 |
Status | | Need SDM to correct point mutation | |
Part | hEF1a:Heavy | hEF1a:Light | UAS:mKate |
Status | | | |
hEF1a:CD79A-XnTCSXc-Gal4VP16
3TCS3 | | 9TCS3 | | 15TCS3 |
| | | | |
| 6TCS6 | 9TCS6 | 12TCS6 | |
| | no colonies | | |
3TCS9 | 6TCS9 | 9TCS9 | 12TCS9 | 15TCS9 |
| | | | |
| 6TCS12 | 9TCS12 | 12TCS12 | |
| | | | |
3TCS15 | | 9TCS15 | | 15TCS15 |
| | | | |
...
50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3tcs15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA50ng
Dox 1 nM anti IgM x uM | WELL 12 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9tcs9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 13 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9tcs9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA50ng
Dox 1 nM anti IgM x uM | WELL 14 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15tcs3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 15 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15tcs3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA50ng
Dox 1 nM anti IgM x uM | WELL 16 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15tcs15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 17 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15tcs15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-3-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM
| | | WELL 18 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng Dox 1 nM | WELL 19 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 20 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng Dox 1 nM | WELL 21 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 22 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng Dox 1 nM | WELL 23 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 24 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 25 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA50ng
Dox 1 nM anti IgM x uM | WELL 26 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 27 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-6-tTEVp 50ng Dummy DNA50ng
Dox 1 nM anti IgM x uM | | |
Plate 3 WELL 28 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 29 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 30 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 31 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 32 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 33 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 34 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 35 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 36 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 37 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 38 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 39 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 40 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 41 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 42 HEK293 50ng hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 43 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 44 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 45 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-9-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | | | | | | |
Plate 4 WELL 46 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 47 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 48 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 49 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-6TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 50 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 51 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 52 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM | WELL 53 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS6-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 54 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 55 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-12TCS12-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-12-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | | | WELL 56 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3TCS3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 57 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3TCS3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 58 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3TCS15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 59 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-3TCS15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 60 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 61 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-9TCS9-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 62 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15TCS3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 63 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15TCS3-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | WELL 64 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15TCS15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM
| WELL 65 HEK293 hEF1a:rtTA 50ng hEF1a:MabH 50ng hEF1a:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng hEF1a:CD79A-15TCS15-Gal4VP16 50ng hEF1a:CD79B 50ng TRE:Syk-15-tTEVp 50ng Dummy DNA 50ng
Dox 1 nM anti IgM x uM | | |
|
Results
Discussion
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