Team:Freiburg/Notebook/lab activation

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Effector - Activation

18.04.13

E001: Digest

Digest of pKM006 with AatII + NheI-HF.

Loading sheme: Marker (didn't work) - Digest of pKM006 - Failed PCR product - Failed PCR product.

Agarose gel

Figure 1: Gel pic 1 (Digest pKM006)

20.04.13

E001: MiniPrep of Traffo(pKM602)

E001: Test digest pKM602 with XmnI, EcoRI

E001: Agarose gel

Checking test digest of pKM602

Agarose gel

Figure 1: Gel pic 2 (Test-Digest pKM602)

Sending first attempt for sequencing --> correct

30.04.13

E008-E011: Digest

Digest of pKM006 with AatII and NheI-HF.

E008-E011: Agarose gel

Agarose gel

Figure 1: Gel pic 3 (Digest pKM006)

E008-E011: Ligation

Ligation of pKM006 with Oligos:

E008: oKM519/520

E009: oKM521/522

E010: oKM523/524

E011: oKM525/526

03.05.13

E008-E011: Miniprep pKM608, pKM609, pKM610, pKM611

E008-E011: Test digest of pKM608-pKM611

Test Digest with pKM608-pKM611 with XmnI, EcoRI.

E008-E011: Agarose gel

Loading sheme: Marker - 1-6 (pKM608) - 1-6 (pKM609) - 1-6 (pKM610) - 1-6 (pKM611)

Figure 1: Gel pic 4 (TestDigest pKM608-pKM611)

Sending for Sequencing:

pKM608_4

pKM609_1

pKM610_1

pKM611_2

All attempts were correct!

05.06.13

E002: Agarose gel

Loading sheme: Marker - Digest pX334a - Marker

Figure 1: Gel pic 5 (Digest pX554a)

06.06.13

New primers arrived (oKMxx1, oKMxx2, oKMxx3)

E002: PCR 1

F2-3 (Cas9 D10A;H840) with oKMxx1/oKMxx2

Size: 1947 bp

E002: PCR 2

pKM018 with oKMxx3/oKM505 for getting VP16

Size: 712 bp

E002: Agarose gel

Loading sheme: Marker - PCR1 (Cas9 D10A;H840) 1-4 - PCR2 (VP16) 1-4 - Marker

Figure 1: Gel pic 6 (PCR 1__PCR 2)

E002: Digest

Digest of PCR1 product (Cas9) with SacI and BamHI: Size: 1868 bp

Digest of PCR2 product (VP16) with BamHI and NotI: Size: 674 bp

07.06.13

E002: Agarose gel

Loading sheme: Marker - Digest Cas9 - Digest VP16 - Marker

Figure 1: Gel pic 7 (Digest of PCR1 (Cas9) and digest of PCR2 (VP16)

E002: Ligation

Ligation with Cas9 (correct overhangs), VP16 (correct overhangs) and digested pX334a (SacI, NotI)

E002: Traffo

Transformation with Ligation attempt.

09.06.13

E002: MiniPrep of pKM600

pKM600_1 - pKM600_12

E002: Testdigest of pKM600

Test digest of pKM600_1 - pKM600_12 with SacI-HF

E002: Agarose gel

Loading sheme: Marker - Test digest pKM600_1 - pKM600_12

Figure 1: Gel pic 8 Test digest of pKM600_1 - pKM600_12 with SacI-HF

19.06.13

Transfection

HEK293T cells were co-transfected with the following plasmid combination

75000 cells/well

Transfection sheme

Figure 1: Transfection sheme with HEK293T cells

27.06.13

SEAP Assay 3

Same conditions as SEAP Assay on 20.06.13

Results of SEAP Assays

Results SEAP Assay

Figure 1: SEAP result with EMXI crRNA

Figure 1: SEAP result with four different VEGFA crRNAs

09. September

Retrafo of Plasmids for the Midiprep

Following plasmids are needed for activation and repression repeats:
• pRSet
• pKM600
• pKM604
• pKM605
• pKM606
• pKM607
• pKM603
• pIG2017
• pIG2013
• pSAM200

10. September

Midiprep

Plasmids were prepped with the Midiprep kit of Promega according to the manufacturer's protocol.

Seeding of cells

4 24-well plates were seeded with 65,000 cells per well

11. September

Transfection

1. 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
4. Solution was spread drop-wise to the cells in the dish

Medium change

13. September

Preparation for analyses

• Supernatant was collected for SEAP measurement.
• Cells were frozen at - 80 °C for later on Western blot and luminescence measurement.

14. September

SEAP measurement

• Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.
• Supernatant was diluted in cell culture medium 1:4 (25 µl supernatant + 75 µl medium).
• 80 µl of diluted supernatant was given to 100 µl of SEAP buffer.
• After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm.

16. September

Cell lysis

• 250 µl of lysis buffer (containing Tris-HCl, EGTA, MgSO₄, DTT and protease inhibitor) were applied to the thawn cells of each well.
• Incubation on ice for 10 min.
• Centrifugation at 15,000 g for 4 min.

Renilla luminescence measurement

• 80 µl of supernatant were pipetted in a white 96 well plate.
• Measurement of luminescence (every 2 min for 30 min) immediately after addition of 20 µl Renilla substrate in PBS.

SEAP activity normalized to Renilla expression The value of TetR-VP16 is 64,6 which is too high to be shown.

The different SEAP plasmids showed very differing background levels of SEAP expression. When Cas9-VP16 without a crRNA was cotransfected there was a reduction of SEAP expression in two cases, whereas in all other samples the SEAP level stayed unaltered. An activation in SEAP expression in comparison to the cells that were not transfected with Cas9-VP16 was only detectable for the EMX1 target sequence (5 fold).
SEAP activation with TetR-VP16 was much stronger, most probably because there are 13 binding sites for TetR-VP16, but only one for Cas9-VP16.

17. September

Protein precipitation

As the cell lysis needed to be done with a high amount of lysis buffer, proteins are too much diluted for western blotting. Thus, they were precipitated:

• The remaining lysates of the triplikates (~ 80 µl) were put together in a new epi.
• Addition of TCA to a final concentration of 10 % and 1 µg BSA.
• Incubation for 30 min on ice.
• Centrifugation for 30 min at full speed and 4 °C.
• Removal of supernatant and addition of 500 µl acetone.
• Incubation on ice over night.
• Centrifugation for 15 min at full speed and 4 °C.
• Removal of acetone and nair drying for 20 min.
• Addition of 20 µl 2x SDS-loading dye and 0.5 µl Tris-HCl (pH = 8.8).
• Heating at 95 °C for 5 min.

18. September

SDS-gel run

SDS-gel was loaded with 18 µl of each sample. A voltage of 80 V was applied till the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.

Western Blotting

• Activation of PVDF-membrane for 10 min in methanol.
• Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.
• Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).
• 200 mA were applied for 1.5 h.

Antibody treatment I

• Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.
• Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
• 3 x washing with TBS-T for 5 min, each.
• Incubation with anti mouse antibody for 1 h.
• 4 x washing with TBS-T for 10 min, each.
• Measurement of luminescence after addition of ECL I + ECL II solution.
• Air drying of the membrane for further antibody treatments.

19. September

Seeding of cells

3 24-well plates were seeded with 65,000 cells per well.

Antibody treatment II

• Reactivation in methanol.
• 1 x washing with TBS-T for 5 min.
• Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 30 min.
• Incubation with anti beta-actin antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
• 3 x washing with TBS-T for 5 min, each.
• Incubation with anti mouse antibody for 1 h.
• 4 x washing with TBS-T for 10 min, each.
• Measurement of luminescence after addition of ECL I + ECL II solution (500 µl each).

Western blot In every well that was transfected with Cas9-VP16 there was more or less the same expression of this fusion protein.

20. September

Transfection

1. 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
4. Solution was spread drop-wise to the cells in the dish

Ratio of DNA amount (mass):
reporter plasmid (SEAP) : effector plasmid (Cas9) : RNA plasmid
1 : 4 : 4

Medium change

22. September

Medium removal

• Supernatant was collected for SEAP measurement.
• Cells were frozen at - 80 °C for later on Western blotting.

Preparation for SEAP measurement

Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.

23. September

SEAP measurement

• Supernatant of wells containing CMV:SEAP was diluted in cell culture medium 1:10 (10 µl supernatant + 90 µl medium).
• 80 µl of (diluted) supernatant was given to 100 µl of SEAP buffer.
• After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm.

SEAP activity [U/L] pKM600: Cas9-VP16 (not in iGEM standard); PhyB: negative control; all other Cas9 constucts are in iGEM standard (with the indicated promoter); bars represent three biological copies when there is an error bar (mean with standard deviation) or one when there is not.

In comparison to the negative control the non standardized constructs repress the SEAP expression only slightly, whereas there is a strong repression by the standardized constructs, when driven by a CMV promoter. The repression of Cas9-KRAB seems to be a little stronger than the effect of Cas9 alone. But just like in the last experiment, the repression of Cas9-KRAB with a wrong crRNA is as strong as the repression with the right one. In comparison to the negative control there was a reduction in SEAP expression when Cas-VP16 was cotransfected.

Seeding of cells

4 24-well plates were seeded with 65,000 cells per well

24. September

Repeat of seeding of cells

4 24-well plates were seeded with 65,000 cells per well again, as the cell density of the first seeding was too low.

25. September

Transfection

1. 40 µl Opti-MEM + 0.75 µg of the DNA of interest were mixed in a 1.5 ml Eppi.
2. Addition of 2.25 µl PEI-solution, vortexing for 10 s and incubation for at least 15 min at RT
3. Solution was spread drop-wise to the cells in the dish

Ratio of DNA amount (mass):
reporter plasmid (SEAP) : effector plasmid (Cas9) : RNA plasmid
1 : 4 : 4
Additionally a plasmid coding for Renilla luciferase was cotransfected (5 % of DNA amount)

26. September

Medium change

Cell lysis of transfection of 20. September

• 80 µl of RIPA lysis buffer were applied to the thawn cells of each well.
• Incubation on ice for 10 min.
• Sonification 3 x 30 s at maximum.
• Centrifugation at 10,000 g for 10 min.
• 40 µl of supernatant were mixed with 10 µl 5 x SDS sample buffer.
• Heating for 5 min at 95 °C.

SDS-gel run

SDS-gel was loaded with 40 µl of each sample. A voltage of 80 V was applied till the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.

Western Blotting

• Activation of PVDF-membrane for 10 min in methanol.
• Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.
• Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).
• 200 mA were applied for 1.5 h.

Antibody treatment I

• Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.
• Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
• 3 x washing with TBS-T for 5 min, each.
• Incubation with anti mouse antibody for 1 h.
• 4 x washing with TBS-T for 10 min, each.
• Measurement of luminescence after addition of ECL I + ECL II solution.
• Air drying of the membrane for further antibody treatments.

Western blot of the SEAP assay from 23.09. dCas-VP16 was expressed with each tested promoter, but strongest with CAG.

27.09.13

Medium removal

• Supernatant was collected for SEAP measurement 48 h after transfection.
• Cells were frozen at - 80 °C for later on Western blotting.

28.09.13

SEAP measurement

• Collected supernatant and fresh cell culture medium was heated at 65 °C for 30 min, then centrifuged at 1250 g for 1 min.
• Supernatant of wells containing CMV:SEAP was diluted in cell culture medium 1:15 (6.7 µl supernatant + 93.3 µl medium).
• 80 µl of (diluted) supernatant was given to 100 µl of SEAP buffer.
• After addition of 20 µl pNPP (substrate of SEAP) the absorbance of the mix was measured every minute for 2 h at 405 nm.

Cell lysis for lyciferase measurement and western blot

• 250 µl of lysis buffer (containing Tris-HCl, EGTA, MgSO₄, DTT and protease inhibitor) were applied to the thawn cells of each well.
• Incubation on ice for at least 10 min.
• Centrifugation at 2,200 g for 30 min.

Luciferase luminescence measurement

• 80 µl of supernatant were pipetted in a white 96 well plate.
• Measurement of luminescence (every 2 min for 15 min) immediately after addition of 20 µl Renilla substrate in PBS.

SEAP-Assay: Results All different tested loci could activate the SEAP-gene expression. Cas-VP16 under the control of the CMV promotor as well as the SV40 promotor worked fine. Using more than one target locus simultaniously increases gene activation.

29.09.13

Protein precipitation

As the cell lysis needed to be done with a high amount of lysis buffer, proteins are too much diluted for western blotting. Thus, they were precipitated:

• 340 µl of acetone was added to 85 µl of the remaining lysates of one well per triplicate.
• Incubation at - 20 °C for 1:15 h.
• Centrifugation for 5 min at 10,000 g and 4 °C.
• Removal of acetone and air drying for 5 min.
• Addition of 25 µl 2x SDS-loading dye.
• Heating at 95 °C for 5 min.

SDS-gel run

SDS-gel was loaded with 20 µl of each sample. A voltage of 80 V was applied untill the loading dye bands reached the border between collection and separation gel, then the voltage was increased to 120 V.

Western Blotting

• Activation of PVDF-membrane for 10 min in methanol.
• Short incubation of Whatman papers and the PVDF-membrane in transfer buffer.
• Loading of western blot apperature: (-) - Whatman paper - SDS-gel - PVDF-membrane - Whatman paper - (+).
• 200 mA were applied for 1.5 h.

Antibody treatment I

• Incubation of PVDF membrane in blocking buffer (4 % milkpowder in TBS-T) for 1 h.
• Incubation with anti HA antibody (in 2 % milkpowder in TBS-T) over night at 4 °C.
• 3 x washing with TBS-T for 5 min, each.
• Incubation with anti mouse antibody for 1 h.
• 4 x washing with TBS-T for 10 min, each.
• Measurement of luminescence after addition of ECL I + ECL II solution.
• Air drying of the membrane for further antibody treatments.

Western blot of the SEAP assay from 28.09. Western blotting did not work well. Maybe lysis due to relative long incubation time on Renilla lysis buffer destroyed cells to much beforehand. So maybe only degradation products are visible.