Cloning

Cloning - May

Cloning - June

Cloning - July

Cloning - August

Cloning - September

Cloning - October

Viral Vectors

Viral Vectors - May

2014/05/21

Transfection/ Virus production

For virus production Phoenix cells (producer cell line) were splitted (well separated) on 100mm plates. At 70% cell density cells were transfected using polyethylenimine.

• remove medium and refill with 5 ml new completed growth medium (DMEM)
• 600 µl transfection mastermix was prepared (8 µg pMIG IRES EGFP, 24 µl PEI, rest OptiMEM)
• mastermix was incubated 15 min and carefully drop on the plates

Plates were incubated at 37°C. The supernatant after 24 was removed and refilled with 5 ml new DMEM, the supernatant was collected after 48 h (refilled with 5 ml DMEM) as well as 72 h.

2014/05/25

Transduction mouse cells

NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP.

• 500 µl of supernatant was removed
• 1 µl Polybrene was added (10mg/ml)
• 500 µl virus supernatant was added (sterile filtered)
• incubation at 37°C for 6h
• cell supernatant was replaced with fresh DMEM
• transduction was repeated once

Pictures could be made after 48 h of incubation.

Viral Vectors - June

2014/06/20

Thawing of eukaryotic cells

New Phoenix cell stocks were thawed:

• cryotube was thawed at 37°C water bath until almost defrosted
• stock was filled in 9 ml warm completed growth medium and centrifuged at 900 rpm for 2 min
• medium was removed and refilled with 10 ml warm completed growth medium
• cells were seeded on 100 mm plates

Testing optimal cell density of mouse fibroblasts

NIH 3T3 have a really fast growth so that we tested the optimal cell number for seeding NIH 3T3 for having around 60% cell density on the next day. NIH 3T3 cells grow very fast; therefore we have tested the optimal seeding cell number to obtain 60% cell density on the next day. Results indicate that the optimal cell number is 1 &ndash 1.5x10^5 cells per well ( = 0.5 – 0.75 cells/ml)

2014/06/22

Transfection/ Virus production

Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (2 x 100mm plate)

2014/06/24

Transfection/ Virus production

Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (5 x 100mm plate)

2014/06/27

Thawing new HEK 293 cells

(protocol: 2014/06/20)

Transfection CHO cells with receptor

Transfection of CHO cells with SLC7a1 (for later transduction with virus). Medium was changed after 5 h. Cells were incubated for 24 h before viral transduction with MuLV IRES EGFP, medium change after 16 h.

2014/06/27

Transduction mouse cells (different incubation times)

NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP and incubated for 8, 16, 24 and 2 x 8 hours. Virus was taken from different supernatants (an older one and a newer one) to see, if it makes any difference. Cells were infected with supernatant (500µl viral supernatant, 500µl completed growth medium + 1µl Polybrene/ml) harvested at different time points. Results indicate that there was no difference between older and newer virus; best results were given with an infection time of 2 x 8 hours.

For testing, if centrifugation brings better transduction efficiencies, mouse cells were infected with the different viral supernatants and centrifuged for 45 min, 1800 rpm, 32°C. In two wells it was tested if the double amount of Polybrene brings better transduction efficiencies. However, we found out that cells were death after centrifugation.

2014/06/30

Transfection CHO cells with receptor

CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

Transfection/ Virusproduction

Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21).

Viral Vectors - July

2014/07/03

Transfection CHO cells with receptor

CHO cells were transfected with the receptor (for later transduction). Medium was removed and filled with 2 ml new medium per well. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C. This time there were no results du to high density of cells during transduction.

Freezing (cryopreservation) of eukaryotic cells

Phoenix cells were frozen at -80°C.

• removal of medium and washing with cold PBS
• addition of 1 ml 0,05% Trypsin per plate, incubation for 1-2 min)
• stopping of reaction with 5 ml DMEM (with FCS)
• centrifugation (5 min, 900 rpm)
• removal of supernatant and resuspension in 2 ml FCS (+10% DMSO)
• quick transfer in steril cryotube (1ml per tube) and quick freezing in -80°C

2014/07/06

Transfection CHO cells with receptor

CHO cells were seeded on cover slips and transfected with the receptor (for later transduction). Due to the fact that cells must be in growth phase during transduction with virus the cell density was set to 40%. Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

2014/07/08

Phoenix cells were transfected with pMIG IRES EGFP (protocol: 2014/05/21)

2014/07/10

Transfection of CHO cells with receptor

CHO cells were transfected with the receptor (for later transduction). Medium was changed after 5 h and cells were transduced with MuLV IRES EGFP. This experiment gave no results.

2014/07/10

Fixation of cells on cover slips

CHO cells (transfected with SLC7a1; 2014/07/04) were fixed on cover slips

• Medium was removed and cells were washed with PBS
• Appropriate amount of 4% PFA/PBS was added (200µl on 24W) and incubated for 10 min on ice
• PFA was removed and plate was washed with PBS
• Cover slips were fixed with Mowiol on slides

2014/07/11

Transfection of HEK cells with receptor

HEK 293 cells were transfected with the receptor (for later transduction). Medium was changed after 5 h. Cells were transduced with MuLV IRES EGFP after 24 h of incubation at 37°C.

2014/07/14

Transfection/ Virus production

2014/07/17

Transduction of mouse cells

Different volumes of virus supernatant were added to mouse cells (on 24W plate, 70% density) and analyzed by FACS (green), Microscopy (yellow) and Western Blot (blue) after 48 h.

green: anaylsis with flow cytometry

yellow: fixation with PFA on cover slips

• Removal of medium
• Washing with cold PBS
• Adding of 400 µl PFA and incubation for 10 min on ice, another 10 min at RT
• Incubation of cover slips for 10 sec in DAPI solution (1:5000 in water)
• Washing in water
• Mounting with Mowiol on slides

Cells detach from the cover slip, therefore a coating is necessary e.g. with Poly-L-Lysine  no results (better use poly-lysine for better grip of cells on cover slip)

blue: preparation for Western Blot via RIPA Lysis (as positive control for anti-CAT1 antibody)

• Removal of medium
• Washing with ice cold PBS
• Addition of 100 µl RIPA Buffer (completed with Phosphatase-Inhibitor-Mix)
• Incubation 10 min on ice
• Removal of cells with tip and transfer into Eppendorf tube
• Incubation for 10 min on ice
• Centrifugation for 5 min 10000 x g
• Transfer of 60 µl supernatant in new tube
• Addition of 15 µl 5 x SDS loading dye (with β-Mercaptoethanol)
• Cooking for 10 min at 95°C or for 15 min at 72°C
• Freezing at -24°C

Transfection of CHO cells with receptor

CHO cells growing in completed HTS medium (K1) were compared to CHO cells growing in completed DMEM medium. Cells were transfected with the receptor. Afterwards both kinds of CHO cells were infected with MuLV IRES EGFP and analyzyd using flow cytometry investigate which cells are better for transfection and transduction. Medium was changed after 5 h. Cells were incubated for 24 h at 37°C.

2014/07/21

QR-code on 96W plate

HEK cells were transfected with the blue light system with SEAP as reporter and seeded on a 96W plate for pattern generation.

• Transfection of HEK293 in suspension (2 x 10^5 cells/ml, 100µl transfection mix/ml) with PKM292, PKM297 and PKM084 (1,5 µg DNA/ml cell suspension)
• Incubation for 5 h at 37°C and seeding on a 96W plate (with photo mask) afterwards (120µl/well)
• After 24 h of incubation at 37°C cells were illuminated with blue light for 5 h.
• Again after 24 h of incubation a SEAP assay was made directly in the plate:
• 30 min incubation of plate at 65°C
• addition of 100µl SEAP buffer into each well (ca. 90µl medium was left in each well)
• before measurement 20 µl substrate was added

Transfection/ Virus production

Phoenix cells were transfected with pMIG IRES EGFP and pMIG EGFP(protocol: 2014/05/21)

2014/07/31

Improvement of Transduction

Transduction of NIH 3T3 cells with two different viral supernatants via three different methods.

• 1.2 µl Polybrene adding directly to 1 ml DMEM on cells and adding 1 ml viral supernatant afterwards
• 2.addition of 1 µl Polybrene to 1 ml viral supernatant and addition of the mixture to 1 ml DMEM on the cells
• 3.addition of 2 µl Polybrene to 1 ml viral supernatant and addition of the mixture to 1 ml DMEM on the cells
• Incubation for 48 h at 37°C

Transduction of NIH3T3 cells with two different viral supernatants (MuLV IRES EGFP or MuLV EGFP) with either 1 or 2 µl Polybrene.

• 1. 1 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant
• 2. 2 µl Polybrene added directly to 1 ml DMEM on cells, addition of 1 ml virus supernatant
• 3. 2µl Polybrene was added to 1 ml viral supernatant; mixture was added to 1 ml DMEM

Centrifugation at 37°C for 45 min at 400 RPM. Results indicate that cells do not like to be centrifuged.

Testing difference in transfection efficiency of Phoenix and HEK cells/ negative control for MuLV

Phoenix cells and HEK cells were tested for there transfection capacity and compared. In addition, it was tested that the virus cannot infect Phoenix nor HEK cells. So both kinds of cells were transfected with PHB308 (mCherry, 3 µg/well) and in parallel transduced with MuLV IRES EGFP (1 ml/well + 2 µl Polybrene).

Viral Vectors - August

2014/08/01

Generation of a GFP mouse cell line

For testing whether MuLV can stable transfer genes into cells, a stable mouse cell line using this virus was generated.

Therefore two 100mm plates were transduced with 3 ml virus supernatant (MuLV IRES EGFP) and splitted as usual. Cells were sorted with a cell sorter. Analysis via FACS happened before and after sorting. The analysis was repeated after several rounds of splitting.

2014/08/03

Testing different transfection methods with different cells

For optimizing transfection in different cell lines transfection methods and different concentrations of the transfection mixtures were compared. The experiment was done with mouse cells (NIH3T3), hamster cells (CHO) and human cells (Phoenix). As transfection reagents lipofectamin and PEI were used in different concentrations.

Transfection with Lipofectamin (for 3 wells):

• (solutions A) 50 µl OptMEM was mixed with either:
• 1. 1 µl Lipofectamin + 1,5 µl PHB308
• 2. 2,5 µl Lipofectamin + 1,5 µl PHB308 or
• 3. 4 µl Lipofectamin + 1,5 µl PHB308
• incubation for 25 min at RT
• (solution B) 150 µl OptiMEM was mixed with 1,5 µl PHB308 (2,5 µg/µl) and 4 µl Plut Reagent
• incubation for 15 min
• solutions A (1-3) were then mixed with 50 µl of solution B and incubated for 5 min at RT
• 100 µl of transfection solution were added to each well
• no medium changing, incubation at 37°C for 24 h

Transfection with PEI (for 1 well):

• 0,2 µl PHB308 was mixed with 40 µl OptiMEM and DNA, for each well another concentration of DNA was added:
• 1. 1,5 µl PEI
• 2. 3 µl PEI
• 3. 5 µl PEI
• Incubation for (optimal) 10,8 min
• addition of 40 µL solution to each well
• medium changing after 5 h of incubation at 37°C, incubation for 24 h at 37°C

2014/08/06

Transfection of HEK cells with receptor

HEK293 were transfected with the receptor plus mCherry to see, if the virus infect only cells expressing the receptor (the two plasmids should infect same cells). In parallel cells were transfected with the blue light and the red light positive control (PMZ422 and PSAM200) and the receptor inducible by the light systems. After 24 h cells (expressing the receptor) were infected with MuLV IRES EGFP.

Transfection HEK and Pheonix cells with receptor (transfection while seeding)

To avoid a too high cell density for transduction cells were transfected while seeding. The PEI transfection mix was given to the HEK or Phoenix cell suspension (1,5 x 10^5 c/ml, 100 µl mix per 1 ml cells) and seeded on a 12W plate. Results indicated that PEI was too toxic for cells during this method.

2014/08/09

Virus dilution

Virus supernatant was diluted with fresh DMEM before transduction. Cells were analyzed after 48 h.

• 0,5 ml virus + 0,5 ml DMEM
• 0,4 ml virus + 0,6 ml DMEM
• 0,3 ml virus + 0,7 ml DMEM
• 0,2 ml virus + 0,8 ml DMEM
• 0,1 ml virus + 0, 9 ml DMEM

2014/08/15

Testing different receptor constructs

HEK293 cells were transfectec with p14rz_004 (labeled with HA-tag), p14ls_006 (labeled with HA-tag and mCherry) and the original receptor plasmid pQCXIN (containing SLC7A1).

Viral Vectors - September

2014/09/01

QR-code on 96W plate

HEK cells were transfected with the blue light system with SEAP as reporter and seeded on a 96W plate for pattern generation.

• Transfection of HEK293 in suspension (2 x 10^5 cells/ml, 100µl transfection mix/ml) with PKM292, PKM297 and PKM084 (1,5 µg DNA/ml cell suspension)
• Incubation for 5 h at 37°C and seeding on a 96W plate (with photo mask) afterwards (120µl/well)
• After 24 h of incubation at 37°C cells were illuminated with blue light for 5 h.
• Again after 24 h of incubation a SEAP assay was made directly in the plate:
• 30 min incubation of plate at 65°C
• addition of 100µl SEAP buffer into each well (ca. 90µl medium was left in each well)
• before measurement 20 µl substrate was added

2014/09/03

Virus Half life time

800 µl aliquots of virus supernatants (MuLV IRES EGFP) were incubated for different times at 37°C in the incubator. After incubation cells were infected with the supernatants (250 ml supernatant/ 250 ml medium + 0,5 µl Polybrene). After 48 h cells were anaysed by FACS.

Experiment: receptor life time after splitting

Two 6W of HEK293 cells were transfected with rz006 (receptor labled with mCherry and HA-tag). After each round of splitting cells were analysed by Western blot. Therefore each round one well was splitted 1:2 on two new wells and one well was lysed by Ripa lysis. Afterwards, each lysed sample was frozen in -20°C until use.

Two new cells lines

New cells lines were thawed:

• MCF-7 human breast cancer cells
• A-547 human lung epithel carcinoma

Rezeptorexpression time

HEK293 cells on 16 x 35mm plates were transfected with rz006 (receptor labeled with mCherry and HA-tag). At several time points after transfection cells were lysed with RIPA buffer and analyzed with Western Blot. Samples were frozen at -20°C until analysis.

We quantified the expression of the receptor after transfection of HEK-293T cells with different concentrations of receptor DNA by Western blotting. 35 mm plates with HEK293T cells were infected with 0.6 to 5.4 µg receptor DNA labeled with HA-tag (p14rz_006). Cells were lysed with RIPA buffer 24 hours after transfection and analyzed by Western blot using an HA-probe antibody.

2014/09/05

Generation of a GFP mouse cell line

For testing whether MuLV can stable transfer genes into cells, a stable mouse cell line using this virus was generated.

Therefore two 100mm plates were transduced with 3 ml virus supernatant (MuLV IRES EGFP) and splitted as usual. Cells were sorted with a cell sorter. Analysis via FACS happened before and after sorting. The analysis was repeated after several rounds of splitting.

2014/09/09

Specificity of MuLV

We tested, if MuLV was specific for murine cell lines and was not able to infect cell lines not containing mCAT-1. So we incubated different human cell lines with MuLV EGFP regarding EGFP expression after infection.

2014/09/11

Testing different pMIG constructs

We tested the expression strength of EGFP in pMIG constructs containing only the marker in comparison to constructs containing the target gene upstream of an IRES and EGFP as a marker.

2014/09/20

Confocal images of HEK293T cells transduced for receptor localization

HEK293T cells were transfected with mCAT-1-mCherry (p14rz_005). 4x10^5 cells/ ml were incubated with PEI transfection mix and receptor DNA (1,5 µg/ml) for 5 hours at 37°C and washed by centrifugation (900 RPM, 2 min, 22°C) afterwards. The pellet was resuspend in completed growth medium and 200 µl cell suspension (1,5 x 10^5 cells/ ml) were seeded on a 24 well plate. Plates were prepared with cover slips covered with gelantine before. After 24 hours of incubation cells were fixed with PFA for confocal microscopy.

2014/09/26

Transfection/ Virus production (stained virus)

Phoenix cells were stained with DiD, a membrane staining dye.

• Resuspension of 2 µl dye (5mM) in 5 ml OptiMEM
• Removing of medium from five 100mm Pheonix plates and addition of 1 ml staining solution per well
• Incubation for 15 min at 37°C
• Washing of plates two times with DMEM (incubation time between each washing step was at least 10 min
• Transfection of cells with pMIG-mKO, pMIG-BFP2, pMIG-EGFP and pMIG-mKate
• Medium changing after 24 h of incubation at 37°C and refill with 4 ml new DMEM
• Harvest after 48 h and 72 h post transfection

2014/09/28

Virusintegration time in mouse cells

NIH3t3 cells (mouse fibroblasts) were infected with MuLV IRES EGFP at different time points. Directly after the latest infection the cells were splitted on new plates. After 48 h cells were analyzed via FACS.

QR-code on 24W plate (with virus)

For testing the functionality of our system and the functionality of the virus with SEAP, cells were transfected with the blue light system plus light inducible receptor (labled with mCherry) and later infected with MuLV SEAP or MuLV CMV SEAP. Both viruses were compared. In addition, cells were infected with MuLV EGFP as control. Several negative controls were added as well as unilluminated cells expressing the light system for testing leaky expression. Cells were incubated in dark.

• Transfection of cells with eather the blue light system plus receptor (PKM292, PKM297, ls003) or the receptor (rz006 p2a mCherry).
• Cells were transfected via PEI in suspension (3 x 10^5 cells/ml, 100 µl transfection mix/ml, 1,5 µg DNA/ml). Cells were incubated for 5 h at 37°C before centrifugation (900 RPM, 3 min, 24°C). Cells were resuspended with fresh DMEM to a final concentration of 1,5 x 10^5 cells/ml) and seeded (0,5 ml/well).
• Cells were incubated for 24 h at 37°C and the illuminated with blue light for 1 h (not the dark control).
• Cells were infected with 250 µl virus supernatant (+0,5 µl Polybrene) per well. Medium was changed after 4 h of incubation at 37°C.
• After 24 h and after 48 h of incubation 200 µl supernatant was taken and used for SEAP assay as described before.

Since, SEAP is already produced by Phoenix cells during generation of viral particles, SEAP was detected in every negative control.

Transduction of mouse cells on cover slips, different colours

Mouse cells (on 25 cm coverslips, covered with polylysine) were infected with MuLV-mKO, MuLV-EGFP, MuLV-BFP2, MuLV-mKate (half DMEM, half virus supernatant) and incubated at 37°C for 48 h. Medium was changed after 16 h of incubation. Afterward cells were analyzed via fluorescence microscopy.

2014/09/29

QR-code with virus on 96W plate

• Cells were transfected in suspension with the blue light system plus receptor (PKM292, PKM297, ls003, 3 x 10^5 cells/ml, 100 µl transfection mix/ml, 1.5 µg DNA/ml). Cells were incubated for 5 h at 37°C before centrifugation (900 RPM, 3 min, 24°C). Cells were resuspended with fresh DMEM to a final concentration of 1.5 x 10^5 cells/ml and seeded (0,1 ml/well).
• After 15 h of incubation cells were illuminated with blue light for 1 h.
• Cells were incubated for 24 h and afterwards infected with MuLV CMV SEAP. Medium was changed after 4 h of infection.
• After 24 h the supernatant was analyzed via SEAP assay as described before.

Viral Vectors - October

Light-System

Light System - June

2014/06/16

Transfection Red light system in CHO cells

We transfected the lightsystem (pKM022) + mCherry (pKM078) & lightsystem (pkMpKM022) + receptor (pMW:SLC7A1)

• we used 4 x 100 mm dishes: 2 x for 660 nm and 2 x for 740 nm illumination (one of each for negative control)
• transfection with red light system (pKM022) and receptor (SLC7A1) or light induced mCherry (pKM078)
• transfection mix was incubated for 20 min and 1 ml added to each dish
• medium was changed after 4-5h
• cells incubated for 20 h

2014/06/17

PCB addition

• 50 mg PCB was dissolved in 2 ml DMSO - every step was performed in the dark!
• c(PCB) = 4.39x10^-5 M = 43,9 mM
• 14,08 µl PCB solution was diluted in 40 ml DMEM
• c(PCB, diluted) = 15 mM
• the culture medium of the transfected cells was replaced by 10 ml prewarmed PCB-DMEM solution
• Three plates were placed for one hour in the 660 nm lightbox (negative control, pKM078, pMW)
• Three plates were placed for one hour in the 740 nm lightbox (negative control, pKM078, pMW)
• after illuminations the lightboxes were incubated at 37 °C for 23 h

2014/06/18

Lysis of cells with RIPA buffer

• For samples transfected with the receptor cells were lysed with RIPA buffer
• Cells were washed with ice-cold PBS
• Cells were lysed with 4,43 ml ice-cold RIPA buffer (completed with phosphatase inhibitor mix) and incubated for 10 min
• Cells were detached from the plate with a inverted pipette tip and transferred into Eppis
• Eppis were vortexed and incubated on ice for 10 min
• Eppis were centrifuged for 5 min at 10.000 x g
• 60 µl direct-lysate with were mixed with 15 µl 5x SDS-Dye and heated for 10 min at 85 °C
• Lysates were stored at - 24 °C

Fixation with PFA

• Cells transfected with pKM078 (mCherry) illuminated at 740 nm, 600 nm; negative control lighted at 660 nm were washed with 30 ml ice-cold PBS
• 3 ml ice-cold PFA-Solution (4%) was added and 10 min incubated on ice
• PFA was replaced with 7 ml PBS and stored in the freezer

Experiment did not work.

2014/06/22

Transfection Red light system in CHO cells

• for each transfection condition an 1.5 ml eppi was prepared with:
• 50 µl Opti-MEM
• plasmid DNA (0.75 µg in total)
• both were mixed by vortexing
• 2.5 µl PEI solution was added
• mixture was directly vortexed for 10 sec
• the solution was incubated at room temperature for 15 min
• the precipitate (50 µl per well) was carefully added to the CHO cells
• the mixture was incubated in the cell incubator
• after 5 h: the medium was changed to prewarmed DMEM
• the CHO cells were incubated for 20 h

	pKM078/pKM006	pKM022	pSAM200	pMIG	Opti-MEM (to 200 µl)	PEI
1) mCherry for leaky expression	58 µl pKM078	/	/	5.2 µl	136,8 µl	10 µl
2) pKM078 -PCB	58 µl	4.5 µl	/	1.7 µl	135.8 µl	10 µl
3) pKM078 +PCB	58 µl	4.5 µl	/	1.7 µl	135.8 µl		10 µl
4) constitutive control	58 µl	/	19 µl	1.7 µl	138.4 µl	10 µl
5) transfection control	/	/	/	7 µl	193 µl	10 µl
6) SEAP for leaky expression	1.3 µl pKM006	/	/	5.2 µl	193.5 µl	10 µl
7) pKM 006 +PCB	1.3 µl	4.5 µl	/	1.7 µl	194.2 µl	10 µl

2014/06/23

PCB addidtion

To activate the red light system PCB was added after 20 hours of incubation

• 3 sample mixtures were prepared and the medium in the wells with 0.5 ml of one of the mixtures replaced:
• 10 ml DMEM - for samples 1,4,5 and 6
• 3.5 ml DMEM + 1.3 µl PCB solution - samples 3 and 7
• 3.5 ml DMEM + 1.3 µl DMSO - samples 2
• (DMSO was diluted 1:8000 in methanol:HCL (95:5 vol/vol))

2014/06/29

Transfection of receptor (SLC7A1) in CHO cells)

• A 6W plate was prepared:
• 1st well: negative control
• 2nd well: transfection control - pEGFP_C1 (3 µg)
• 3rd well: transfection control - pEGFP_C1 (3 µg)
• 4th well: SLC7A1 (3 µg)
• 5th well: SLC7A1 (3 µg)
• 6th well: SLC7A1 (2 µg)

Cells were lysed with RIPA buffer 24 hours after incubation at 37°C

• cells were washed with 0.5 ml PBS
• 200 µl RIPA buffer was added to the well
• cells were scraped off and transferred into a tube
• cells were incubated on ice on 10 min
• the mix was vortexed and incubated on ice for 10 min
• lysates were centrifuged for 5 min at 10.000 x g
• 170 µl direct-lysate of each sample was added to 42.5 µl 5x SDS loading-dye
• samples were incubated for 10 min at 95°C
lysates were stored at -26°C

transfection of the red light system into CHO cells

• 2x 24 well plates were trancfected, one plate with cover slips and one plates without cover slips
• 24 well plate with cover slips:
• 5 wells: 0.5 µg pKM022 + 0.25 µg pKM078 + PCB
• 5 wells: 0.5 µg pKM022 + 0.25 µg pKM078 - pCB
• 5 wells: 0.5 µg pSAM200 + 0.25 µg pKM078
• 3 wells: 0.5 µg EGFP_C1 + 0.25 µg pKM078
• 3 wells: 0.75 µg EGFP_C1
• 3 wells: negatve control
• 24 well plate without cover slips:
• 5 wells: 0.5 µg pKM022 + 0.25 µg pKM006 + PCB
• 5 wells: 0.5 µg pKM022 + 0.25 µg pKM006 - pCB
• 5 wells: 0.5 µg pSAM200 + 0.25 µg pKM006
• 3 wells: 0.5 µg EGFP_C1 + 0.25 µg pKM006
• 3 wells: 0.75 µg EGFP_C1
• 3 wells: negatve control
• transfection mastermixes were incubated for 15 min
• after 5h: medium exchange in both 24 well plates

2014/06/30

PCB addition

• after 21h: medium was changed (0.5 ml per well)
• 5 wells (0.5 µg pKM022 + 0.25 µg pKM006 + PCB): DMEM medium supplemented with 15 µM PCB
• 5 wells (0.5 µg pKM022 + 0.25 µg pKM078 + PCB): DMEM medium supplemented with 15 µM PCB
• 5 wells (0.5 µg pKM022 + 0.25 µg pKM006 - PCB): DMEM medium supplemented with DMSO
• 5 wells (0.5 µg pKM022 + 0.25 µg pKM006 - PCB): DMEM medium supplemented with DMSO
• in all other wells: medium was changed to prewarmed DMEM medium
• both 24 well plates were illuminated for 24 h in the 660 nm lightbox at 37°C

Cells were lysed with RIPA buffer after 24 hours. Lysates were incubated with SDS loading dye for 10 min at 95°C and stored at -24°C.

Light System - July

2014/07/07

Measurement of LED band

• intensity of our LED band light box: ca. 6 µmol/(m^2*s)
• intensity of both light boxes from other laboratory (AG Weber): 20 µmol/(m^2*s)

2014/07/08

Transfection of red light system in CHO cells/ comparison of different light boxes

• transfection scheme in 24 well:

A: 0.5 µg pKM022 + 0.25 µg pKM006	A: 0.5 µg pKM022 + 0.25 µg pKM006	A: 0.5 µg pKM022 + 0.25 µg pKM006	B: 0.5 µg pSAM200 + 0.25 µg pKM006	B: 0.5 µg pSAM200 + 0.25 µg pKM006	B: 0.5 µg pSAM200 + 0.25 µg pKM006
C: 0.5 µg pKM022 + 0.25 µg pKM006	C: 0.5 µg pKM022 + 0.25 µg pKM006	C: 0.5 µg pKM022 + 0.25 µg pKM006	D: 0.5 µg empty vector + 0.25 µg pKM006	D: 0.5 µg empty vector + 0.25 µg pKM006	D: 0.5 µg empty vector + 0.25 µg pKM006
E: 0.75 µg pMIG	E: 0.75 µg pMIG	E: 0.75 µg pMIG	F: 0.5 µg pKM022 + 0.25 µg078 (on cover slides)	F: 0.5 µg pKM022 + 0.25 µg078 (on cover slides)	F: 0.5 µg pKM022 + 0.25 µg078 (on cover slides)
G: pHB362 + pKM082	G: pHB362 + pKM082	G: pHB362 + pKM082	H: pHB362 + pKM082	H: pHB362 + pKM082	H: pHB362 + pKM082

• medium was changed 5h after transfection

2014/07/09

PCB addition

To activate the red light system PCB was added after 20 hours of incubation

• Two solution-mixes were prepared:
• 1) 2x (15 µl our PCB solution + 37.5 ml DMEM)
• 2) 2.8 µl Weber's BCB solution + 7 ml DMEM)
• the medium in all wells was aspirated
• 0.5 ml solution-mix 1 was added to wells labeled with A, B, D, F, G, H
• 0.5 ml solution-mix 2 was added to wells labelled with C
• plates were first incubated in the dark for 1h then illuminated for 24h

After 24 hours of illumination 200 µl supernatant of samples A, B, C, D, E, G, H was transferred in 96W plates. The plate was stored at -24°C before measurement with SEAP-assay. Samples F were fixed with PFA and stored at 4°C.

2014/07/11

Preparation of SEAP buffer

• 2.25 g homoarginine + 0.048 g MgCl2 diluted in 250 ml ddH2O
• 105 ml diethanolamine was added
• pH value was set to 9.8 with HCL solution
• filled up to 500 ml with ddH2O

SEAP assay Red light system/ comparison different light boxes

• 96 well plates were sealed and incubaed at 65°C for 45 min
• 1 min centrifuged at 1258 g
• 100 µl SEAP buffer was prepared in each well of a 96 (flat-bottom) well plate
• of the centrifguged plates 80 µl supernatant per well were added to the the 100 µl SEAP buffer
• each well was completed with 20 µl pNPP (substrate)
• spectroscopic measurement every minute for 1 hour at 405 nm







2014/07/11

Transfection of CHO cells with the red light system.

1: 0.5 µg pKM022 + 0.25 µg pKM006	1: 0.5 µg pKM022 + 0.25 µg pKM006	1: 0.5 µg pKM022 + 0.25 µg pKM006	2: 0.5 µg pSAM200 + 0.25 µg pKM006	2: 0.5 µg pSAM200 + 0.25 µg pKM006	2: 0.5 µg pSAM200 + 0.25 µg pKM006
4: 0.75 µg pMIG-IRE-GFP	4: 0.75 µg pMIG-IRES-GFP	4: 0.75 µg pMIG-IRES-GFP	3: 0.5 µg pMIG-IRES-GFP + 0.25 µg pK Retrieved from "http://2014.igem.org/Team:Freiburg/Content/Notebook/Labjournal" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers