Progress
| Cloning | Transfection | Induction | Cytometry | Data Analysis |
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title
Plate 1
WELL 1
HEK293
WELL 2
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
TRE:Syk-15-tTEVp
Dox x uM
WELL 3
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
hEF1a:mKate
TRE:Syk-15-tTEVp
Dox x uM
WELL 4
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
TRE:Syk-15-tTEVp
Dox x uM
WELL 5
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
TRE:Syk
Dox x uM
WELL 6
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
TRE:Syk-15-tTEVp
Dox x uM
WELL 7
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
hEF1a:CD79A
hEF1a:CD79B
TRE:Syk-15-tTEVp
hEF1a:GAl4VP16
Dox x uM
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 BsaI site in Gal4 |
Part | hEF1a:Heavy | hEF1a:Light | UAS:mKate |
Status |
| 3TCS3 | 9TCS3 | 15TCS3 | ||
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
hEF1a:CD79A-3tcs3-Gal4VP16
hEF1a:CD79B
TRE:Syk-3-tTEVp
dox x uM
WELL 26
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
hEF1a:CD79A-3tcs3-Gal4VP16
hEF1a:CD79B3WELL 27
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
hEF1a:CD79A-3tcs15-Gal4VP16
hEF1a:CD79B
TRE:Syk-3-tTEVp
dox x uM
WELL 28
dox x uM
anti IgM x uM
UAS:mKate3tcs15Gal4VP163doxanti IgM x uM
WELL 29
:eBFPUAS9tcs9Gal4VP163dox
WELL 30
UAS9tcs9Gal4VP163doxanti IgM x uM
WELL 31
-3tcs15-Gal4VP163dox
WELL 32
3tcs15Gal4VP16-3-tTEVpdoxanti IgM x uM
WELL 33
-15tcs15-Gal4VP163 doxWELL 34
HEK293
15tcs153 x uManti IgMWELL 37
6TCS66WELL 38
HEK293
hEF1a:MabH
hEF1a:MabL
hEF1a:Lyn
UAS:mKate
hEF1a:CD79A-6TCS6-Gal4VP16
hEF1a:CD79B
TRE:Syk-6-tTEVp
dox x uM
anti IgM x uM
WELL 39UAS:mKate6TCS12Gal4VP166dox40eBFPUAS:6TCS12Gal4VP166dox
anti IgM x uM
41UAS-9TCS9-Gal4VP166dox429TCS9Gal4VP16-6-tTEVp
dox x uM
anti IgM 43-12TCSX6-Gal4VP166dox44-12TCS6-Gal4VP166dox
anti IgM x uM
WELL 45
HEK293
hEF1a:MabH
hEF1a:12TCS126 4612TCS126
Plate 3
WELL 49
539TCS39 549TCS39 559TCS69 569TCS69 579TCS99 589TCS99 599TCS129 609TCS129 619TCS159 629TCS159
6312TCS99 6412TCS99 6515TCS99 6615TCS99
Plate 4 736TCS612 746TCS612 756TCS1212 766TCS1212 779TCS912 789TCS912 7912TCS612 8012TCS612 8112TCS1212 8212TCS1212
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 BsaI site in Gal4 |
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
-VE control (background activation)
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hEF1a:CD79B
TRE:Syk
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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)
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Experimental Conditions
- anti IgM
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anti IgM x uM
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Results
Discussion