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Team:Freiburg/Notebook/lab activation
From 2013.igem.org
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Effector - Activation | Effector - Activation | ||
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<h2>09. September</h2> | <h2>09. September</h2> | ||
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<td> <b>Western blot </b><br> | <td> <b>Western blot </b><br> | ||
| - | In every well that was transfected with Cas9-VP16 there was more or less the same expression of | + | In every well that was transfected with Cas9-VP16 there was more or less the same expression of this fusion protein. |
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<td> <b>SEAP activity [U/L]</b><br> | <td> <b>SEAP activity [U/L]</b><br> | ||
| - | + | 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. | |
</td> | </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
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 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. | ||
</div> | </div> | ||
Revision as of 12:07, 27 September 2013
Effector - Activation
September
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
- 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
- 0.75 µg of the DNA of interest were prepaired in another Eppi .
- Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
- Solution was spread drop-wise to the cells in the dish
Medium change
Medium was changed after 5 h.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. |
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
- 40 µl Opti-MEM + 2.25 µl PEI-solution were mixed in a 1.5 ml Eppi.
- 0.75 µg of the DNA of interest were prepaired in another Eppi .
- Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
- 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
Medium was changed after 4.5 h.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. |
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
- 40 µl Opti-MEM + 0.75 µg of the DNA of interest were mixed in a 1.5 ml Eppi.
- Addition of 2.25 µl PEI-solution, vortexing for 10 s and incubation for at least 15 min at RT
- 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
Medium was changed after 8 h.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.
