Page History

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

hEF1a:CD79B

TRE:Syk-15-tTEVp

Dox x uM

HEK293

hEF1a:MabH

hEF1a:MabL

hEF1a:Lyn

hEF1a:CD79A-XnTCSXc-Gal4VP16

UAS:mKate

hEF1a:CD79A

hEF1a:CD79B

TRE:Syk-15-tTEVp

hEF1a:GAl4VP16

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

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

...

...

hEF1a:MabH

hEF1a:MabL

hEF1a:Lyn

UAS:mKate

hEF1a:CD79A-3tcs3-Gal4VP16

hEF1a:CD79B

TRE:Syk-3-tTEVp

dox x uM

HEK293

hEF1a:MabH

hEF1a:MabL

hEF1a:Lyn

UAS:mKate

hEF1a:CD79A-3tcs3-Gal4VP16

hEF1a:CD79B

TRE:Syk-3-tTEVp

dox x uM

anti IgM x uM

...

HEK293

hEF1a:MabH

hEF1a:MabL

hEF1a:Lyn

UAS:mKate

hEF1a:CD79A-3tcs15-Gal4VP16

hEF1a:CD79B

TRE:Syk-3-tTEVp

dox x uM

3TCS9	6TCS9	9TCS9	12TCS9	15TCS9

	6TCS12	9TCS12	12TCS12

3TCS15		9TCS15		15TCS15

TRE:Syk-X-tTEVp

3	6	9	12	15

Procedure

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

UAS:mKate

hEF1a:CD79A-3tcs15-Gal4VP16

hEF1a:CD79B

TRE:Syk-3-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:

...

mKate

hEF1a:CD79A-

...

15TCS15-

...

G4VP16

hEF1a:CD79B

TRE:Syk-

...

15-tTEVp

...

Dox x uM

anti IgM 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

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

dox x uM

anti IgM x uM

...

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

HEK293

- 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

hEF1a:MabH

hEF1a:MabL

hEF1a:Lyn

UAS:mKate

hEF1a:CD79A-XnTCSXx-Gal4VP16

hEF1a:CD79B

TRE:Syk-Xs-tTEVp

...

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

Expand

title	Plate map

Plate 1

WELL 1

HEK293

WELL 2

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

...

hEF1a:CD79A-

...

15TCS15-

...

G4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

15-tTEVp

...

50ng

Dummy DNA 100ng

Dox 1 nM

WELL 3

dox x uM

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

hEF1a

...

:mKate 50ng

hEF1a:CD79A-

...

15TCS15-

...

G4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

15-tTEVp

...

50ng

Dummy DNA 100ng

Dox 1 nM

WELL 4

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

...

hEF1a:mKate 50ng

hEF1a:CD79A

...

50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

15-tTEVp

...

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 5

dox x uM

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

15TCS15-

...

G4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk

...

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 6

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A

...

50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

15-tTEVp

...

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 7

dox x uM

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A

...

50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

15-tTEVp 50ng

hEF1a:GAl4VP16

...

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

50ng

Dox 1 nM

Plate 2

WELL 8

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

3tcs3-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

3-tTEVp

...

dox x uM

...

HEK293

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 9

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

3tcs15-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

3-tTEVp

...

dox x uM

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 11

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

9tcs9-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

3-tTEVp

...

dox x uM

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 13

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

15tcs3-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

3-tTEVp

...

dox x uM

50ng

Dummy DNA 50ng

Dox 1 nM

WELL 15

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

15tcs15-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

3-tTEVp 50ng

dox x uM

Dummy DNA 50ng

Dox 1 nM

WELL 17

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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

dox x uM

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

6TCS6-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

6-tTEVp 50ng

Dummy DNA 50ng

dox x uM

Dox 1 nM

WELL 19

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

6TCS12-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

6-tTEVp 50ng

Dummy DNA 50ng

dox x uM

Dox 1 nM

WELL 21

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

9TCS9-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

6-tTEVp 50ng

Dummy DNA 50ng

dox x uM

Dox 1 nM

WELL 23

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 50ng

UAS:mKate 50ng

hEF1a:CD79A-

...

12TCS6-Gal4VP16 50ng

hEF1a:CD79B 50ng

TRE:Syk-

...

6-tTEVp 50ng

dox x uM

Dummy DNA 50ng

Dox 1 nM

WELL 25

...

HEK293

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:CD79A-XnTCSXc-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.

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

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)

...

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

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

hEF1a:GAl4VP16

Dox x uM

...

50ng

hEF1a:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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:MabH 50ng

hEF1a:MabL 50ng

hEF1a:Lyn 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

...

Experimental Conditions

...

- 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