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Expand
titleTest 1
Expand
titleDescription

Tests the effectiveness of a Syk-TEV cofactor in the tango system with no transfected Lyn, only possible endogenous Lyn.

Expand
titlePurpose

Capstone Experiment to test Syk-TEV without Lyn.

Expand
titleParts Needed

hEF1a: Gmab H

hEF1a: Gmab L

hEF1a: CD79A

hEF1a: CD79B

TRE: Syk-TEV

hEF1a: rtTA

UAS: mKate

Expand
titleSetup

Well 1

HEK293

Well 2

HEK293

Well 3

HEK293

Well 4

HEK293

Well 5 

HEK293

Well 6 

HEK293

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

Expand
titleExpected Results

We expect the tango cleavage system to have virtually no difference between active and inactive receptors because the BCR requires (question) (actually, some papers say that it doesn't?) Lyn to recruit Syk.

Expand
titleProtocol

Day1

Seed the cells on a plastic plate using splitting cells and seeding plates protocol

Day2

Transfect the cells using either the lipo2k protocol

Day3

add a dox ladder

Day4

do standard cytometry prep

Expand
titleMicroscopy Data

 

Expand
titleCytometry Data

 

Expand
titleAnalysis

 

Expand
titleTest 2
Expand
titleDescription

Tests the effectiveness of a Syk-TEV cofactor in the tango system with no transfected Lyn, only possible endogenous Lyn.

Expand
titlePurpose

Capstone Experiment to test Syk-TEV without Lyn.

Expand
titleParts Needed

hEF1a: Gmab H

hEF1a: Gmab L

hEF1a: CD79A

hEF1a: CD79B

TRE: Syk-TEV

hEF1a: rtTA

UAS: mKate

Expand
titleSetup

Well 1

HEK293

Well 2

HEK293

Well 3

HEK293

Well 4

HEK293

Well 5 

HEK293

Well 6 

HEK293

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

 

 

 

Well 1Well 2Well 3Well 4Well 5Well 6

untransfected

(calibration)

eBFP bleed through controlleaky mKate control

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

HEK293

 

 HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug Dummy

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: mKate

 

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B

100 ug UAS: mKate

100 ug Dummy

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B-TCS-G4

100 ug UAS: mKate

100 ug Dummy

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B-TCS-G4

100 ug UAS: mKate

100 ug Dummy

Well 7Well 8Well 9Well 10Well 11Well 12

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

10 nM dox

 

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A-TCS-G4

50 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

200 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A-TCS-G4

25 ug CD79B

100 ug : Syk-TEVp

100 ug UAS:mKate

300 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A-TCS-G4

12.5 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

350 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A-TCS-G4

6.25 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

375 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A-TCS-G4

3.125 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

387.5 ug Dummy

10 nM dox

Well 13Well 14Well 15Well 16Well 17Well 18

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A

50 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

200 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A

25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

300 ug Dummy

10nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A

12.5 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

350 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A

6.25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

375 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A

3.125 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

387.5 ug Dummy

10 nM dox

 

Well 19Well 20Well 21Well 22Well 23Well 24

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

10 nM dox

 

100 ugHEK293

100 ughEF1a: eBFP

100 ughEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A-TCS-G4

50 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

200 ug Dummy

10 nM dox

 

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A-TCS-G4

25 ug CD79B-TCS-G4

100 ugTRE: Syk-TEVp

100 ug UAS:mKate

300 ug Dummy

10nM dox

 

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A-TCS-G4

12.5 ug CD79B-TCS-G4

100 ugTRE: Syk-TEVp

100 ugUAS:mKate

350 ug Dummy

10 nM dox

 

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A-TCS-G4

6.25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

375 ug Dummy

10 nM dox

 

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A-TCS-G4

3.125 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

387.5 ug Dummy

10 nM dox

Expand
titleExpected Results

We expect the tango cleavage system to have virtually no difference between active and inactive receptors because the BCR requires (question) (actually, some papers say that it doesn't?) Lyn to recruit Syk.

Expand
titleProtocol

Day1

Seed the cells on a plastic plate using splitting cells and seeding plates protocol

Day2

Transfect the cells using either the lipo2k protocol

Day3

add a dox ladder

Day4

do standard cytometry prep

Expand
titleMicroscopy Data

 

Expand
titleCytometry Data

 

Expand
titleAnalysis

 

 

 

Test 3

 

...

Well 1

...

Well 2

...

Well 3

...

Well 4

...

Well 5

...

Well 6

...

untransfected

(calibration)

...

TEVp control

(nonspecific G4 cleavage)

...

TEVp control

(nonspecific G4 cleavage)

...

TEVp control

(nonspecific G4 cleavage)

...

HEK293

 

...

 HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug Lyn

 

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP

100 ug Lyn

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B

100 ug UAS: eYFP

 

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B-TCS-G4

100 ug UAS: eYFP

 

...

100 ug HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B-TCS-G4

100 ug UAS: eYFP

 

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

 

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A-TCS-G4

50 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A-TCS-G4

25 ug CD79B

100 ug : Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A-TCS-G4

12.5 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A-TCS-G4

6.25 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A-TCS-G4

3.125 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A

50 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A

25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A

12.5 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A

6.25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

...

HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A

3.125 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

 

...

100 ug HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B-TCS-G4

...

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

 

...

100 ugHEK293

100 ughEF1a: mKate

100 ughEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A-TCS-G4

50 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

...

10 nM dox

 

...

100 ug HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

...

25 ug GmabL

25 ug CD79A-TCS-G4

25 ug CD79B-TCS-G4

100 ugTRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10nM dox

 

...

100 ug HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A-TCS-G4

12.5 ug CD79B-TCS-G4

100 ugTRE: Syk-TEVp

100 ugUAS: eYFP
100 ug Lyn

10 nM dox

 

...

100 ug HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A-TCS-G4

6.25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

10 nM dox

 

100 ug HEK293

100 ug hEF1a: mKate

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A-TCS-G4

3.125 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS: eYFP
100 ug Lyn

...