Team:Freiburg/Notebook/lab light

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29.07.2013

Plasmids that we need as templates were transformed.

30.07.2013

Primers

Primers arrived today. Primers were diluted 1:10.

Minipreps

Transformed bacteria from yesterday with our template plasmids were minipreped.

• pKM018: 83 ng/µl
• pKM220: 216 ng/µl
• pKM115: 177 ng/µl
• pKM006: 118 ng/µl
• pSAM200: 213 ng/µl
• pSW42: 134 ng/µl
• pSW45: 243 ng/µl
• pIG2004: 212 ng/µl
• T405: 213 ng/µl

Oligo Annealing of tetO crRNA

• Oligos tetO13 1. CC and tetO13 2. CC were annealed
• Therefore 20 µM diluted Primes were heated on 98° C for 4 minutes and then cooled down.

PCR list, program and composition

• aIG4102: CMV Promotor from template T405 - Primers oIG4100F+R - 589 bp
• aIG4103: Cas9 from template pIG2004 - Primers oIG4101F+R - 4170 bp
• aIG4104: PIF and backbone from template pKM022- Primers oIG4102F+R - 3287 bp
• aIG4105: KRAB from template pIG2004 - Primers oIG4103F+R - 390 bp
• aIG4106: PhyB and backbone from template pKM018 - Primers oIG4104F+R - 4922 bp
• aIG4107: UVR8 and backbone from template pKM168 - Primers oIG4105F+R - 3921 bp
• aIG4108: COP1 and backbone frome template pKM115 - Primers oIG4106F+R - 3972 bp
• aIG4109: CRY2 from template pSW42 - Primers oIG4107F+R - 1591 bp
• aIG4110: Backbone from template pKM018 - Primers oIG4108F+R - 2924 bp
• aIG4111: Backbone with VP16 from template pKM018 - Primers oIG4109F+R - 3282 bp
• aIG4112: CIB from template pSW45 - Primers oIG4110F+R - 593 bp
• aIG4113: Backbone from template pKM018 - Primers oIG4111F+R - 2881 bp
• aIG4114: CIB from template pSW45 - Primers !! oIG4110F+oIG4112R - 638 bp

PCR results

• aIG4102: 60° C - WORKED - GelExtraction: 87,9ng/ul
• aIG4103: 60° C - 1 of 4 WORKED, Annealing Temperature lower? - GelExtraction: 50,5ng/ul Try 56°!
• aIG4104: 60° C - DIDN'T WORK!!! Try with 58° - Was done, has to be tested on gel.
• aIG4105: 60° C - WORKED - GelExtraction: 70,1ng/ul I and 57,7ng/ul II
• aIG4106: 60° C - Was done, has to be tested on gel.
• aIG4107: 60° C - ONLY SLIGHT BAND. TRY AGAIN!
• aIG4108: 60° C - WRONG AMPLIFICATED BAND. Try again with different temperature!
• aIG4109: 60° C - WORKED! - GelExtraction: 176,4ng/ul
• aIG4110: 60° C - Was done, has to be tested on gel.
• aIG4111: 60° C - WORKED! - GelExtraction: 84,4ng/ul
• aIG4112: 60° C - Wasn't done yet.
• aIG4113: 60° C - Was done, has to be tested on gel.
• aIG4114: 60° C - Wan't done yet.

31.07.13

PCR results

• aIG4103: 54°-64° C - Everything WORKED!
• aIG4104: 58° C - Gave three bands, upper band is supposed to be right. Was confirmed on a second gel with charging the gel only with a small amount of DNA.
• aIG4106: 60° C - Didn't work. Try temperature gradient.
• aIG4107: 54°-64° C - Everything Worked! But not very strong bands.
• aIG4108: 54°-64° C - Didn't Work. Try seperate PCRs.
• aIG4110: 60° C - Didn't work. Tried temperature gradient 54°-59° C - Worked with 55° and better with every additional ° C.
• aIG4112: 60° C - Worked!
• aIG4113: 60° C - Worked!
• aIG4114: 60° C - Worked!

01.08.13

PCR results

• aIG4104: Was gelexed and size was confirmed on a gel with a smaller amount of DNA. - Worked!
• aIG4106: Temperature gradient 54° - 59° C and excess of reverse primer oIG4104R - Worked!
• aIG4108: Seperate PCRs were tried out. Therefore the PCR was performed with template and at a time only one primer. After these PCRs (1x with forward Primer (58° C) and 1x with reverse Primer (62° C)) both columns were mixed and another PCR was performed. - Worked, but two bands appeared, upper band is considered to be the right fragment.

Gibsons

Fragments are mixed together to a total volume of 5µl and added to 15µl Gibson Master- Mix.

pIG4101: aIG4104- 0,83µl; aIG4103- 3,96µl; H2O- 0,21µl

pIG4103: aiG4107- 2,12µl; aiG4103- 2,88µl

pIG4105: aIG4110- 1,11µl; aIG4103- 3,51µl

pIG4106: aIG4111- 0,5µl; aIG4112- 0,3µl, H2O- 4,2ul

pIG4107: aIG4113- 0,4µl; aIG4105- 0,35µl; aIG4114- 0,35µl; H2O- 3,9µl

Gibson Mixes were transformed.

Ligation of pIG4100

Digest for pIG4100

Ligation and Transformation of approach pIG4100

After the ligation, 2,5 µl if this approach were transformed into E.coli and scratched out.

02.08.13

Gibson & Ligation results

Colonies could be seen on every plate except for one of the two ligation plates. Clones were picked and scratched out -> 51 Minis

Gibson

pIG4102: aIG4105- 0,4µl; aIG4106- 2,00µl; H2O- 2,6 µl

pIG4104: aiG4108- 3,2µl; aiG4105- 0,5µl; H2O- 1,3µl

Gibson Mixes were transformed.

03.08.13

Gibson results

Colonies could be seen on one of two plates, each. Colonies were scratched out for mini preps. Therefore we had clones for every construct.

Minipreps

51 Minis - concentrations between 50 and 200 ng/µl.

04.08.13

Minipreps

15 Minis.

05.08.13

Test digest

Today we made a test digest session of our 66 minis with following enzymes.

For the sequencing we used GATC Primers: CMV-F for pIG4100 and EBV-RP (binds in pSV40) for all other plasmids.

Insertion of crRNA into RNA Plasmid

1. First RNA Plasmid backbone was digested with BbsI for 3 hours. Enzyme was taken out of - 80° C.
2. Then it was put on a gel and gelexed. Concentration: 50,5ng/ug. Backbone from Hormone- Group: 8,7ng/ul
3. Ligation: Digested backbone from Hormone- Induction and self- digested backbone was used. Molar ratio of backbone : insert - 1 : 3.
4. Mix: Backbone; Insert, 1ul T4- ligase, 2ul T4-ligase-buffer, H2O up to a total volume of 20ul
5. Incubated for 15min at RT
6. Transformed and scratched out.

06.08

Sequencing results

Sequencing results were
positive for pIG4101, pIG4102, pIG4103, pIG4105, pIG4106 and pIG4107
(sequenced with EBV-RP -> SV40 Terminator). pIG4100 didn't have the CMV promotor as an insert (-> new ligation) and pIG4104 didn't contain KRAB, although KRAB worked with pIG4102 (new Primers?)

Re-Trafos and inoculation for midi-preps

The positive Plasmids and already existing plasmids pKM018 and pKM115 were retransformed and put into 150 ml LB with 150 µl Ampicillin and incubated at 37° C over night.

New Primers for pIG4104

New Primers were designed for pIG4104.

Ligation of pIG4100

Ligation of pIG4100 was done again like on 31.07.13, only the ratio of backbone and insert was changed (3 µl backbone and 1,5 µl Insert).

Result of Ligation of RNA Plasmid

There were clones on every plate. They were scratched out for minipreps.

New sequencing

Plasmids were send to GATC with different primers. Cas9 was sequenced completely in pIG4101.7. Other pIG4104 were screened for insertion of KRAB.

7.8.2013

Midipreps of pKM017; pIG4101.7 (?), pIG4102.1, pIG4103, pKM115, pIG4105.5, pIG4106.3, pIG4107.4

All midipreps were successfull

Concentrations:

pKM018: 474 ng/ul

pIG4101: 485 ng/ul

pIG4102: 528 ng /ul

pIG4103: 521 ng/ul

pKM115: 517 ng/ul

pIG4105.1: 492 ng /ul

pIG4106: 567 ng/ul

pIG4107: 476 ng /ul

Minipreps of RNA Plasmid pIG4201 and pIG4202

Minis were send to GATC. 3 of ligation with digested Backbone from Michi and tetO1 and 3 with tetO2. And 3 of ligation with newly digested Backbone from Gabi and tetO1 and 3 with tetO2.

Result of Ligation of pIG4100

There were clones on every plate. They were scratched out for minis.

8.8.2013

Sequencing of RNA Plasmid

1 of the 6 pIG4201 and 4 of the 6 pIG4202 were positiv with inserted crRNA.

Sequencing Order

Sequencing of pIG4107_2 and pIG4107_3 with Primer EBV-RP

Minis of pIG4100

12 Minis were prepped.

Re-Trafo for inoculation for Midi Preps

The following plasmids were retransformed: pIG1000, pIG4201, pIG4202

9.8.2013

Test digest of pIG4100

10.8.2013

Seeding of CHO cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

11.8.2013

Transfection of CHO cells

Protocol for 24- well plates:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

12.8.2013

PCB- treatment of CHO-cells and illumination

Cells are treated with HTS-medium containing 15uM of phycocyanobilin

illumination started at 16.15. with 20uE light- quantity

plate 1 with activating red light (660nm)

plate 2 with inctivating far-red light (740nm), control

construction of pIG4104

pKM115 was digested with BamHI and BssHII for 4h. KRAB was amplified from pIG2004 with oIG4103FII and oIG4103RII

Subsequently the fragments were assembled via Gibson-cloning and transformed into E.coli

12.8.2013

Gibson results

Cell-colonies are picked and plated out anew

A mistake in the cloning strategy was discovered, new primers are designed

13.8.2013

Gibson results

CHO- cells are illuminated

14.8.2013

Seeding of CHO-cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

SEAP-measurements of illuminated cells

The results are all negative except for the positive control (-> see transfection Nr.6 of 11.8.2013)

The interaction with the cri-RNA may be responsible. The experiment is repeated with new target- sites

15.8.2013

Transfection of CHO-cells for red light and UV tests

The wiki- transfection protocoll is used:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

The following pattern is transfected in 24- well plates:

16.8.2013

UV-b and red-/ far red light illumination of transfected cells from 15.8.2013

Cells for red- light illumination are illuminated with 20uE; 660nm or 720nm for 24h. Illumination is started at 14.20

Cells for UV-b light illumination are illiminated with 5uE; 311nm for 24h

17.8.2013

SEAP-measurements taken from illuminated- and untreated cells.

the following protocoll was obeyed for the whole process

200 µl of cell supernatant were transfered form the 24-well plate into one 96-well plate (round bottom)

Incubation for 30 min at 65 °C.

Centrifugation for 1 min at 1250 g.

100 µl of SEAP buffer were filled in the wells of a 96 well plate (flat bottom).

Addition of 80 µl supernatant from the round bottom plate to each well with SEAP buffer.

Addition of 20 µl pNPP (substrate).

96 plate was immediately placed in the blade reader.

Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.

numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013

the system has to be improved, the controls showed the desired effect, but very weak. In the following the expression of Cas9- constructs has to be checked and improved. The tetO cri-RNA showed an insufficient binding pattern. New target sites for the plasmid have to be construct

20.8.2013

Construction of new cri-RNAs

6 new cri-RNAs are designed and ordered at sigma-aldrich. One new cri-RNA is provided by Max Stamnitz. All new cri-RNAs target pKM006!

Cloning of pIG4100; pIG4104; pIG4106; pIG4107

Primers for new cloning- strategies have arrived.

pIG4100: pKM006 is digested and the CMV-promotor from pAcGFP1 is amplified via PCR.

pIG4104: pKM115 is digested with BamHI and BssHII. KRAB is amplified via PCR from pIG2001

pIG4106: pKM018 is digested with EcoRI and NotI. CIB is amplified from pSW45

pIG4107: pIG4102 is digested with EcoRI and NotI. CIB is amplified from pSW45

21.8.2013

Cloning of pIG4100; pIG4104; pIG4106; pIG4107

Products from digestions and PCRs are purified via gel run in 1% agarose and gelexed The digest of pKM115 for pIG4104 has not worked and is repeated executing the same protocol.

Parts for pIG4100, pIG4106, pIG4107 are Gibson cloned and subsequently transformated into competent "TOP10" E.coli- cells

22.8.2013

Cloning of pIG4100; pIG4104; pIG4106; pIG4107

Clones of Gibsons are picked and plated out. Later to be picked and test- digested. For each plasmid 2 samples were sent to GATC for sequencing.

Gibson- cloning of pIG4104 is repeated with new digest and PCR-fragments. The constructs are subsequently transformated into competent "TOP10" E.coli- cells

Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

HEK, HeLa and CHO- cells are seeded in 6-well plates

23.8.2013

Cloning of pIG4104

Colonies are picked and plated out for mini and test- digest on 24.- or 25.8.13

Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

different DNA- concentrations are used for transfection in different cell lines. pIG1000 containing Cas9-VP16 is used. As a already tested sample, pIG8004 containing Cas9-G9a is used as positive- control. The following DNA- concentrations are transfected: 2ug; 3ug; 4ug; 6ug. One sample is tested with and addition of 0,5% DMSO into the medium attempting to boost the expression

The following pattern is transfected

24.8.2013

Oligo annealing

Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.

Ligation of new RNA plasmid

Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

25.8.2013

Result of ligation

Colonies were picked for mini preps.

26.8.2013

Minipreps of targets

Minipreps were sent for sequencing

27.8.2013

Result of sequencing

T2, T5 and T6 were positive.

Seeding of cells for UV light activation experiment

29.8.2013

Transfection of CHOs for UV light activation

30.8.2013

Light start

UV light was started at 15.30 o clock (two plexi glass panes)

31.8.2013

SEAP assay

SEAP assay of UV light activation was performed.

03.9.2013

Oligo annealing

• 5µl Oligo forward 100 µM to get 20 µM concentration
• 5 µl Oligo reverse 100 µM to get 20 µM concentration
• 10 µl NEB buffer 2
• 30 µl ddH2O

Mix was heated to 98° C, Heatblock was turned off and Oligos were cooled down

Digest of pIG3010

• 10 µl pIG3010
• 1 µl BbsI from -80° C
• 5 µl NEB buffer 2.1
• up to 50 µl ddH2O

Digestion was put on a gel and purified (10 ng/µl).

Ligation of Target 3,4 and VEGF and transformation

Digested pIG3010 (10 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

04.9.2013

Result of Ligation

Colonies were picked

Gibson of pIG4104

Wrong primers were used for the last pIG4104 plasmid (was seen with the last sequencing). Therefore we made a new Gibson approach.

05.9.2013

Minipreps of T3, T4 and VEGF (plates) and pIG4104 (fluid culture)

Minipreps were send for sequencing (Primers for RNA Plasmid (in pIG3010) BGH-reverse and EBV-RP for pIG4104.)

06.9.2013

Sequencing results

pIG4104-3 was positive, T3, T4 and VEGF RNA plasmids were negative.

New Ligation of T4 target into standardized RNA plasmid

Procedure was done by standard protocol.

Seeding of CHO cells

4 24-well plates were ceeded with 65000 CHO cells per well.

07.9.2013

Status of CHO cells

CHO cells were all contaminated with bacteria again. Media was plated in a 10 cm dish. Result: No contamination here.

seeding of HEK cells

HEK cells were seeded for UV & BL activation and repression experiments at around 19/19.30. 4 24-well plates and 65000 cells per well.

08.09.2013

Transfection of HEKs for UV & BL activation and repression

HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.

T4 RNA plasmid

6 colonies were scratched out.

09.09.2013

Minis of T4

3 of 6 plasmids were sent for sequencing (1-3).

Light start

Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity number). Dark control is in a light box without light on.

UV light was started at 20.30. Dark control is in the incubator (no UV light in the lab).

10.09.2013

Sequencing results

Sequencing results were positive for Bla Target 4. Bla Targets 2 and 3 were cloned by Philipp who got positive results, too. Plasmids were retransformed and Midis were inoculated.

Seeding of HEK cells.

4 24 well plates were seeded with 65.000 HEK cells per well.

Light stop

Lightning of UV and BL were stopped after about 24 h and supernatants were taken and frozen.

11.09.2013

Midis

Midis of Bla target 2 (pIG4), Bla target 3 (pIG4, Bla target 4 (pIG4), EMX1 (pIG4 and VEGF4 (pIG4) were prepped.

SEAP of UV and BL activation and repression

Activation was okay, repression was in overflow.

Transfection for Activation-VP16 and Repression-KRAB target test row

In order to test which target we should use, we made test rows with different targets and different target combinations. Medium was changed after 5 h.

12.09.2013

SEAP of Activation VP16 test row

Seeding of HEK cells for light activation experiments

13.09.2013

SEAP of Repression KRAB test row and UV and BL repression

Samples had to be diluted 1:10 because the constitutive CMV promoter is very strong. SEAP levels were still in overflow.

Transfection for UV/BL/RL activation

14.09.2013

Light start for UV/BL/RL

15.09.2013

Light stop for UV and BL

SEAP of UV and BL activation

16.09.2013

Light stop for RL

SEAP for RL

18.09.2013

New charge of medium for CHOs

Medium was complemented with FCS, Pen Strep and Gentamicin.

20.09.13

New CHO-K1 & NIH cells

We received CHO-K1 cells from a freshly thawed cell batch. Thank you Shima from AG Ulbrich! These cells were cultivated in DMEM.

We received NIH cells.

24.09.13

Seeding of CHO-K1 (from Toolbox) cells

6 24 well plates were seeded with about 65.000 cells per well.

25.09.13

Transfection for red/blue/UV light induction in CHO-K1 (from Toolbox)

CHO-K1 cells were transfected for red, blue and UV light induced gene activation. Cells that were transfected for UV light were also transfected with Renilla. This was done for normalization, because UV light can kill the cells.

HTS medium was changed after 5 hours.

Seeding of NIH cells

2 24 well plates were seeded with NIH cells (65.000 cells per well).

26.09.13

Transfection for UV light induction in NIH cells

Transfection plan was the same as for CHOs on 25.09.

Light start for red/blue/UV light in CHO-K1 cells (from Toolbox)

1 plate was placed in UV light (311 nm). 1 plate was placed in a red light box (660) after PCB treatment. 1 plate was placed in a far-red light box (740 nm) after PCB treatment. One plate was placed in a blue light box (460 nm).

27.09.13

Light start for UV light in NIH cells

1 plate of transfected NIH cells was placed in UV light.

Light stop for UV (CHO-K1 from toolbox) & blue light (CHO-K1) cells

200 µl of supernatant of each well were removed and heated. After this the 96 well plate was frozen (-20° C). SEAP measurement was done on the next day.

28.09.13

SEAP measurement of UV (CHO-K1 from toolbox) & blue light (CHO-K1) cells

Light stop for red light (CHO-K1 from toolbox) and UV light (NIH)

200 µl of supernatant of each well were removed and heated. SEAP measurement was done according to standard protocol.

29.09.13

Seeding of CHO-K1 from AG Ulbrich and NIH cells

6 24 well plates with CHO-K1 were seeded and 6 24 well plates with NIH cells were seeded.

30.09.13

Transfection of CHO-K1 & NIH cells - red, blue and UV light

Alltogether 12 24 well plates were transfected.

Transfection plans

transfection calculation

01.10.13

Light start

Both transfected cell lines were illuminated: 1 CHO plate & 1 NIH plate with red light 660 nm, 1 CHO plate & 1 NIH plate with blue 465 nm and 1 CHO plate & 1 NIH plate with UV light 311 nm. For every cell line and light system there was a dark control plate (red light's dark control is far red light of 740 nm because the system is reversible)

02.10.13

Light stop

Light was stoped for UV and blue light illumination (after 24 hours) (CHO and NIH cells). 200 µl supernatant were removed from each well for SEAP measurements. SEAP measurement was done according to standard protocol.

03.10.13

Light stop

Light was stoped for red light illumantion. 200 µl supernatant were removed from each well for SEAP measurements. SEAP measurement was done according to standard protocol.