Progress
| Cloning | Transfection | Induction | Cytometry | Data Analysis |
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Plate 1
Plate 2
Plate 3
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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.
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 |
| 3TCS3 | 9TCS3 | 15TCS3 | ||
| 6TCS6 | 9TCS6 | 12TCS6 | ||
| 3TCS9 | 6TCS9 | 9TCS9 | 12TCS9 | 15TCS9 |
| 6TCS12 | 9TCS12 | 12TCS12 | ||
| 3TCS15 | 9TCS15 | 15TCS15 | ||
| 3 | 6 | 9 | 12 | 15 |
Experiment
Controls
| Untransfected | Single Color Blue | Single Color Red | Double Color | -VE control (background activation) | -VE control (background cleavage) | +VE control |
|---|---|---|---|---|---|---|
| HEK293 | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn
hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk-15-tTEVp Dox x uM | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn
hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk-15-tTEVp Dox x uM | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn hEF1a:CD79A-15TCS15-G4VP16 hEF1a:CD79B TRE:Syk-15-tTEVp Dox x uM | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn UAS:mKate hEF1a:CD79A hEF1a:CD79B TRE:Syk-15-tTEVp Dox x uM | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn UAS:mKate hEF1a:CD79A-15TCS15-G4VP16
hEF1a:CD79B TRE:Syk Dox x uM | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn 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 |
|---|---|
HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn UAS:mKate hEF1a:CD79A-XnTCSXc-Gal4VP16 hEF1a:CD79B TRE:Syk-Xs-tTEVp
dox x uM | HEK293 hEF1a:MabH hEF1a:MabL hEF1a:Lyn UAS:mKate hEF1a:CD79A-XnTCSXc-Gal4VP16 hEF1a:CD79B TRE:Syk-Xs-tTEVp dox x uM anti IgM x uM |
| Xs | XnTCSXc |
|---|---|
| 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 |
Results
Discussion