Team:Freiburg/Notebook/lab repression

name	ng/µl
pSAM200	320.1
pKM006	113.3
pKM102	180.0

May

02. May

PCR of pX334 fragments

PCR 1

PCR 2

PCR 3

PCR 4

PCR 5

All samples were stored at -20°C

02. May

Agarose Gelelectrophoreses of PCR fragments No. 1-5

Gel Extraction of PCRs 1-6

name	ng/µl
Fragment No. 1	40.7
Fragment No. 2	53.6
Fragment No. 3	52.6
Fragment No. 4	41.6
Fragment No. 5	40.4

Two bands were respectively pooled on one extraction column, in order to yield higher amount of DNA.

20. May

Gibson Assembly of pIG2004 (Cas9-KRAB)

21. May

Evaluation of Gibson Assembly

Colony PCR Mastermix for Gibson Clones

22. May

Miniprep of suspected pIG2004 Clones 5, 6 and 7

23. May

Evaluation of Gibson Cloning Sequencing Results

PCR of pX334 fragment 6

PCR 1

Agarose Gelelectrophoreses of PCR fragments No. 6

Gel Extraction of PCR 6

name	ng/µl
Fragment No. 6	43.6

Gibson Assembly of pIG2005 (dCas9)

24. May

Digest of pIG2004 (No. 5, 6 and 7) with BbsI

Agarose Gelelectrophoreses of pIG2004 Digests

Gel Extraction of pIG2004 digest

name	ng/µl
pIG2004 (5)	38.0
pIG2004 (6)	17.0

Evaluation of Gibson Assembly of 23. May

27. May

Evaluation of pIG2004 sequencing results (No. 5 and 6)

• H840A mutation was inserted in both cases, leading to catalytic inactivation of Cas9
• a 300 bp gap was inserted within the CAG promoter, probably due to error-prone PCR amplification

04. June

Minipreps of pIG2005 clones striked out on 23. May

07. June

Fixing of the 300 bp CAG promoter gap for Cas9-KRAB

Restriction digest of pIG2004 and pIG2005 - 1

Restriction digest of pX334 - 2

Restriction digest of pX334 - 3

Gel Extraction of digest fragments

name	ng/µl
Cas9-KRAB	26.7
dCas9	5.0
pX334 Backbone	14.4
CAG promoter	5.0

Ligation and transformation of intact pIG2004 and pIG2005

Ligation of pIG2005

Ligation of pIG2004

08. June

Evaluation of Transformations of pIG2005 and pIG2004

• pIG2004 (30 µl strikeout): 24 colonies
• pIG2005 (30 µl strikeout): 15 colonies
• 12 colonies of both approaches were striked out for further sequencing

09. June

Minipreps of pIG2004 and pIG2005

• Colonies No. 2 and 3 of pIG2004 were sent to sequencing, intending to validate (i) KRAB-insertion (ii) H840A conversion and (iii) an intact CAG promoter
• Colonies No. 1 and 4 of pIG2005 were sent to sequencing, intending to validate (i) H840A conversion and (ii) an intact CAG promoter

11. June

Evaluation of Sequencing results of pIG2004 and pIG2005

• pIG2004 clone No. 2 misses the KRAB fragment, but contains the CAG promoter
• pIG2004 clone No. 3 contains KRAB fragment, H840A and the CAG promoter - and will therefore be used for further studies
• pIG2005 clones No. 1 and 4 contain both H840 mutation and the CAG promoter

Restriction digest of pIG2004 and pIG2005 with BbsI

Gel Extraction of digested vectors pIG2004 and pIG2005

name	ng/µl
pIG2004	16.1
pIG2005	18.8

12. June

Oligo annealing of target crRNAs

µl	type
3	Oligo No. 1
3	Oligo No. 2
69	NEB-Buffer 2

• Samples were heated up to 95 degrees for 5 minutes. Gradient cool down to room temperature for 2 hours.
• Pair 1: oIG2010/oIG2011 - EMX1
• Pair 2: oIG2022/oIG2023 - Target 200 bp upstream of SEAP transcription initiation on pKM006
• Pair 3: oIG6008/oIG6009 - CMV

13-20. June

Ligation and transformation of crRNAs with Cas9-KRAB and dCas9

Ligation of pIG2013

µl	type
15	Annealed EMX1
3	oIG2004
2	T4 ligase buffer
1	T4 ligase

Ligation of pIG2017

µl	type
15	Annealed EMX1
3	oIG2005
2	T4 ligase buffer
1	T4 ligase

Ligation of pIG2019

µl	type
15	Annealed SEAP target
3	oIG2004
2	T4 ligase buffer
1	T4 ligase

Ligation of pIG2020

µl	type
15	Annealed SEAP target
3	oIG2005
2	T4 ligase buffer
1	T4 ligase

Ligation of pIG2011

µl	type
15	Annealed CMV target
3	oIG2004
2	T4 ligase buffer
1	T4 ligase

Ligation of pIG2015

µl	type
15	Annealed CMV target
3	oIG2005
2	T4 ligase buffer
1	T4 ligase

Evaluation of transformation results

• Except for pIG2020, colonies were grown in each case
• 6 colonies of pIG2011, pIG2013, pIG2015, pIG2017 and pIG2019 were respectively striked out
• All attempts had to be repeated at least twice, as BbsI digestion often lead to unspecific digestion of direct-repeats next to crRNA insertion spacer.

18-25. June

MIDI-preps of pIG2004, pIG2005, pIG2011, pIG2013, pIG2015, pIG2017, pIG2019 and also pIG9000

25.-27. June

Test of Cas9-KRAB on Western Blots

• Cas9-KRAB and dCas9, both targetting the CMV promoter were respectively co-transfected to HEK 293 cells with another plasmid, encoding CMV driven CNK.
• Both attempts were performed repeatedly, and cell lyses were than to be performed 12, 18, 36 and 42 hours post-transfection.
• Transfections were performed with PEI
• Lyses were performed with modified RIPA buffer
• Biological double attempts were respectively done

Transfection schemes for one 6-well plate

Cas9-KRAB vs. CMV of CNK (2x)

dCas9 vs. CMV of CNK (2x)

CNK negative control (2x)

Cas9-KRAB and dCas9 expression controls on one well

Results

• We expected a stronger decrease in CNK levels, when combining it with Cas9-KRAB than with dCas9
• Cas9-KRAB was able to highly efficiently repress CMV driven CNK expression
• Cas9-KRAB was expressed.

29. June

Test of Cas9-KRAB vs. CMV-driven GFP

• Transfection of pIG6000 (CMV::GFP) in combination with either pIG2011 (Cas9-KRAB + crRNA agaisnt CMV) or Mock DNA
• Cells were seeded previously seeded on 6-well plates
• Incubation for about 36 hours

dCas9 vs. CMV of GFP

01. July

Fluorescence Microscopy Evaluation

• Mean of GFP fluorescence intensity of cells treated with Cas9-KRAB is 1409.4 (SD: 880.6), cells co-transfected with Mock-DNA revealed 2718 (SD: 786.6)
• Results should be of low validity, but are potentially indicative towards a repressive effect of Cas9-KRAB

02. July

MIDI-prep of pIG2005, pIG2019 and pRSET (Mock-DNA)

Failed in each case, for unknown reasons (white salt fall-out after elution from column). Attempt will be repeated on the next day.

03. July

MIDI-prep of pIG2005, pIG2019 and pRSET (Mock-DNA)

04. July

Transfection of pIG2019 in combination with pKM006 (SEAP plasmid) and pSAM200 (SV40::tetR-VP16)

• Biological tetraplicates (each attempt was repeated independently, for four different wells of one 6-well plate
• Negative control: pKM006 (500 ng) + Mock-DNA (2500 ng)
• Positive control: pKM006 (500 ng) + pSAM200 (500 ng) + Mock-DNA (2000 ng)
• Cas9-KRAB test: pKM006 (500 ng) + pSAM200 (500 ng) + pIG2019 (2000 ng)

07. July

SEAP measurement

• 300µl of medium supernatants were removed from each well
• Samples were heated to 65°C for 30 minutes
• Centrifugation at 600 rpm (table top) for 1 minute
• 80µl of each supernatant was transfered to one well of a 96-well plate, technical triplicates for each sample were pipetted
• 100µl 2x SEAP Buffer was added to each well
• 20µl pNPP substrate was added to each well
• Air bubbles were quickly removed
• Measurement was taken with a plate reader, for two consecutive hours with an interval lenght of one minute. Detection wavelength was 405 nm

01. September

Test of Cas9-KRAB vs. CMV-driven GFP, Repetition of the Experiment from 29. June on 24 well plates

dCas9 vs. CMV of GFP

dCas9 vs. CMV of GFP

03. September

Fluorescence Microscopy of KRAB-mediated GFP-repression

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

Retrafo was inoculated into Ampicillin medium (concentration of 1:1000 from Ampicillin and medium).

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

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.

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).

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

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.

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

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.

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.

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.