Team:Freiburg/Notebook/KRAB

From 2013.igem.org

(Difference between revisions)
Line 1,965: Line 1,965:
<tr>
<tr>
<td> pIG2011 (Cas9-KRAB) </td>
<td> pIG2011 (Cas9-KRAB) </td>
 +
<td> 0,46 </td>
 +
</tr>
 +
<tr>
 +
<td> PEI </td>
 +
<td> 1,5 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h4> dCas9 vs. CMV of GFP </h4>
 +
<div id="${1:floatleft}">
 +
<table class="${2:tabelle}">
 +
<tr>
 +
<th> name </th>
 +
<th> &micro;g </th>
 +
</tr>
 +
<tr>
 +
<td> CMV-GFP </td>
 +
<td> 0,04 </td>
 +
</tr>
 +
<tr>
 +
<td> pRSET (Mock-DNA) </td>
<td> 0,46 </td>
<td> 0,46 </td>
</tr>
</tr>

Revision as of 18:02, 12 September 2013

21. April

Minipreps of pKM006, pKM102 & pSAM200

name ng/µl
pSAM200 320.1
pKM006 113.3
pKM102 180.0

02. May

PCR of pX334 fragments

PCR 1

µl type
10 Q5-HF Reaction Buffer
1 pX334
1 oIG2000
1 oIG2029
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 115 sec.
  • Cycles: 18

PCR 2

µl type
10 Q5-HF Reaction Buffer
1 pX334
1 oIG2028
1 oIG2001
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 115 sec.
  • Cycles: 18

PCR 3

µl type
10 Q5-HF Reaction Buffer
1 pX334
1 oIG2002
1 oIG2003
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 115 sec.
  • Cycles: 18

PCR 4

µl type
10 Q5-HF Reaction Buffer
1 pX334
1 oIG2006
1 oIG2007
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 115 sec.
  • Cycles: 18

PCR 5

µl type
10 Q5-HF Reaction Buffer
1 pX334
1 oIG2008
1 oIG2009
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 115 sec.
  • Cycles: 18

All samples were stored at -20°C

02. May

Agarose Gelelectrophoreses of PCR fragments No. 1-5

f.l.t.r.: marker, PCR 1 (1909 bp), PCR 2 (2610 bp), PCR 3 (1645 bp), PCR 4 (2272 bp), PCR 5 (1878 bp)

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

03. May

Fusion PCR of PCR fragments 2-3 and 5-1

Assembly fragment 2-3 (PCR 1)

µl type
10 Q5-HF Reaction Buffer
1 Fragment No. 2
1 Fragment No. 3
4 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 71°C
  • Extension: 105 sec.
  • Cycles: 5

Assembly fragment 2-3 (PCR 2)

  • Addition of 1 µl of oIG2028 and oIG2003 to PCR 1
  • Annealing: 60 °C
  • Extension: 2 min 40 sec
  • Cycles: 16

Assembly fragment 5-1 (PCR 1)

µl type
10 Q5-HF Reaction Buffer
1 Fragment No. 5
1 Fragment No. 1
4 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 71°C
  • Extension: 105 sec.
  • Cycles: 5

Assembly fragment 5-1 (PCR 2)

  • Addition of 1 µl of oIG2008 and oIG2029 to PCR 1
  • Annealing: 60 °C
  • Extension: 2 min 40 sec
  • Cycles: 16

Gel Extraction of Fusion fragments 2-3 and 5-1

name ng/µl
Fragment No. 2-3 (a) 76.6
Fragment No. 2-3 (b) 57.5
Fragment No. 2-3 (c) 106.8
Fragment No. 5-1 23.4

17. May

PCR of the KRAB fragment with a N-terminal linker (GSAGSAG) overhang

PCR 1

µl type
10 Q5-HF Reaction Buffer
1 pKM102
1 oIG2004
1 oIG2005
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 30 sec.
  • Cycles: 18
  • For higher Gel Extraction yields: 4x amount

Agarose Gelelectrophoreses of the KRAB fragment

f.l.t.r.: marker, KRAB at 490 bp (4x)

Gel Extraction of the KRAB fragment

name ng/µl
KRAB (a) 76.0
KRAB (b) 64.7

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)

  • Preparation of 5µl DNA mix, containing all four fragments
  • Melting a 15µl master-mix on ice, until it is ready to use
  • Addition of 5µl of DNA mix to the master mix
  • Immediately put the mix on 50°C and incubate for 1h
  • 3 min on RT
  • 3 min on ice
  • Transformation of 4 µl to TOP10 chemically competent E-coli cells
Preparation scheme for Gibson Assembly fragment mix

21. May

Evaluation of Gibson Assembly

  • Colony growth
  • Colony PCR will be performed with primers within KRAB and Cas9 fragment

Colony PCR Mastermix for Gibson Clones

µl type
34.8 H2O
13.0 Taq Buffer
13.0 dNTPs
1.3 oIG2002
1.3 oIG2005
1.6 Taq Polymerase
5.0 H2O for each reaction, containing picked colony
  • Annealing: 60°C
  • Extension: 68°C
  • Extension: 2 min 10 sec.
  • Cycles: 30
Results of Colony PCR: Clones 5, 6 and 7 were striked out.

22. May

Miniprep of suspected pIG2004 Clones 5, 6 and 7

name ng/µl
pIG2004, No. 5 207.9
pIG2004, No. 6 180.0
pIG2004, No. 7 198.1

Clones were sent to sequencing at GATC

23. May

Evaluation of Gibson Cloning Sequencing Results

oIG0007 sequencing reveals that the KRAB fragment was succesfully fused to Cas9 in each case. Further sequecing will be done to test for correct N-terminal assembly and H840A mutation.

PCR of pX334 fragment 6

PCR 1

µl type
10 Q5-HF Reaction Buffer
1 pX334
1 oIG2002
1 oIG2007
2.5 dNTPs
1.5 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension: 2 min 40 sec.
  • Cycles: 18

Agarose Gelelectrophoreses of PCR fragments No. 6

f.l.t.r.: marker, PCR 6 (3908 bp), empty, PCR 6 (2x)

Gel Extraction of PCR 6

name ng/µl
Fragment No. 6 43.6

Gibson Assembly of pIG2005 (dCas9)

  • Preparation of 5µl DNA mix, containing all four fragments
  • Melting a 15µl master-mix on ice, until it is ready to use
  • Addition of 5µl of DNA mix to the master mix
  • Immediately put the mix on 50°C and incubate for 1h
  • 3 min on RT
  • 3 min on ice
  • Transformation of 4 µl to TOP10 chemically competent E-coli cells
Preparation scheme for Gibson Assembly fragment mix

24. May

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

µl type
Volume DNA
16 pIG2004
10 NEB-Buffer 2.1
2 BbsI
72 H2O
  • Temp.: 37°C
  • Incubation time: 2h

Agarose Gelelectrophoreses of pIG2004 Digests

f.l.t.r.: marker, pX334, empty, digested pIG2004 5 (2x), 6 (2x) and 7 (2x). Upper bands of clone no. 5 and 6 digests were cut out for Gel Extraction.

Gel Extraction of pIG2004 digest

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

Evaluation of Gibson Assembly of 23. May

  • Colony growth
  • Five clones were striked out

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
Very GC-rich part on pIG2004 template (part of the CAG promoter lacks nearly 300 bp after having been amplified via PCR and subsequently Gibson-assembled.

04. June

Minipreps of pIG2005 clones striked out on 23. May

name ng/µl
pIG2005 - No. 1 77.1
pIG2005 - No. 2 68.4
pIG2005 - No. 3 59.6
pIG2005 - No. 4 43.7
pIG2005 - No. 5 61.2

07. June

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

Restriction digest of pIG2004 and pIG2005 - 1

µl type
8 pIG2004 - No. 5 or pIG2005 - No. 1
8 NEB-Buffer 4
2 AgeI-HF
2 NotI-HF
2 SacII
2 BSA
Add to 80µl H2O

Restriction digest of pX334 - 2

µl type
8 pX334 DNA
8 NEB-Buffer 4
2 KpnI-HF
2 NotI-HF
2 SacI-HF
2 BSA
Add to 80µl H2O

Restriction digest of pX334 - 3

µl type
8 pX334 DNA
8 NEB-Buffer 4
2 KpnI-HF
2 AgeI-HF
2 BSA
Add to 80µl H2O
  • Temp.: 37°C
  • Incubation time: 1h
  • Expected fragment lengths: 1 - 4500 bp (Cas9-KRAB), 2 - 4900 bp (Backbone), 3 - 890 bp (CAG promoter)
Top left. Upper band: Cas9-KRAB fragment, Top right. Upper band: dCas9 fragment. Bottom left. Upper band: pX334 Backbone fragment.
Top right. Lowest band (blurred): CAG promoter fragment.

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

µl type
11.2 CAG fragment
4.4 dCas9 fragment
1.4 pX334 backbone
2 T4 ligase buffer
1 T4 ligase

Ligation of pIG2004

µl type
0.8 Cas9-KRAB fragment
14.8 CAG fragment
1.4 pX334 backbone
2 T4 ligase buffer
1 T4 ligase

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

name ng/µl
pIG2004 - No. 1 198.4
pIG2004 - No. 2 203.6
pIG2004 - No. 3 202.0
pIG2004 - No. 4 187.3
pIG2004 - No. 5 195.3
pIG2004 - No. 6 211.2
pIG2004 - No. 7 187.4
pIG2004 - No. 8 201.1
pIG2004 - No. 9 201.7
pIG2004 - No. 10 206.3
pIG2004 - No. 11 212.3
pIG2004 - No. 12 174.6
pIG2005 - No. 1 202.0
pIG2005 - No. 2 195.3
pIG2005 - No. 3 189.9
pIG2005 - No. 4 221.4
pIG2005 - No. 5 205.4
pIG2005 - No. 6 194.4
pIG2005 - No. 7 206.7
pIG2005 - No. 8 206.3
pIG2005 - No. 9 220.4
pIG2005 - No. 10 192.9
pIG2005 - No. 11 188.8
pIG2005 - No. 12 192.1
  • 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

µl type
6 DNA
4 NEB-Buffer 2.1
2 BbsI
Add to 40µl H2O
F.l.t.r.: marker, pIG2004 undigested, pIG2004 digested, pIG2005 undigested, pIG2005 digested (2x)

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

name ng/µl
pIG2004 (Cas9-KRAB) 400
pIG2005 (dCas9) 203
pIG2011 (Cas9-KRAB vs. CMV) 610
pIG2013 (Cas9-KRAB vs. EMX1) 609
pIG2015 (dCas9 vs. CMV) 460
pIG2017 (dCas9 vs. EMX1) 520
pIG2017 (dCas9 vs. EMX1) 520
pIG2019 (Cas9-KRAB vs. SEAP) 430
pIG9000 (Cas9 vs. EMX1) 460
  • MIDIs had to be repeated three times
  • During the first attempt, an unkown white precipitate was obtained - which could not be separated from the desired DNA.
  • Within the second attempt, DNA was not adequately treated during isopropanol treatment (has to be shaken).

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)

name µg
CNK 6
pIG2011 12

dCas9 vs. CMV of CNK (2x)

name µg
CNK 6
pIG2015 12

CNK negative control (2x)

name µg
CNK 6

Cas9-KRAB and dCas9 expression controls on one well

name µg
pIG2004 1
pIG2005 1

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

name µg
CMV-GFP 1
pIG2011 (Cas9-KRAB) 2
PEI 9
Transfection scheme

01. July

Fluorescence Microscopy Evaluation

Fluorescence Images

ImageJ: Cells treated with GFP and with pRSET (Mock-DNA)

Cell Size Intensity
1 208 2920.9
2 146 3945.0
3 238 3064.8
4 166 3699.3
5 238 2071.3
6 192 2219.1
7 172 3819.9
8 228 3235.1
9 247 3510.0
10 146 3213.6
11 228 1977.4
12 224 3436.5
13 224 3436.5
14 136 1872.9
15 140 1611.4

ImageJ: Cells treated with GFP and Cas9-KRAB-DNA

Cell Size Intensity
1 268 2407.6
2 176 3329.2
3 217 1336.1
4 166 1138.5
5 102 2844.8
6 217 936.6
7 148 1236.4
8 214 1032.9
9 177 1566.6
10 96 1461.9
11 228 1977.4
12 938 3371.7
13 102 992.9
14 210 982.1
15 112 1016.6
  • 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)

name ng/µl
pIG2005 (dCas9) 470
pIG2019 (Cas9-KRAB vs. SEAP target) 430
pRSET (Mock DNA) 790

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
SEAP Results

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

name µg
CMV-GFP 0,04
pIG2011 (Cas9-KRAB) 0,46
PEI 1,5

dCas9 vs. CMV of GFP

name µg
CMV-GFP 0,04
pRSET (Mock-DNA) 0,46
PEI 1,5

03. September

Fluorescence Microscopy of KRAB-mediated GFP-repression

SEAP Results

Retrieved from "http://2013.igem.org/Team:Freiburg/Notebook/KRAB"