Team:Freiburg/NotebookDelivery

Lab Journals

Labbook enzymatic activity and sporeshampoo

05.08.2016

plate coated with GFP:

Prepared coating solution: used protocol from “Abeam: direct Elisa coating”. Took 0.78 g Na2CO3, 1.5 g NaHCO3 and dissolved it in 250 ml H2O, pH: 9.5.
Dilute stock solution of GFP (1350µg/ml) in coating buffer for coating concentration of 20 µg/ml.
Coated 93 wells with 50µl coating solution
Incubated for 2 h at room temperature
Washed the wells twice with PBS (V=200 µl)
Blocked it with 5% nonfat dry milk in PBS (2.5 g in 50 ml PBS), blocked coated wells with 200 µl solution.
Incubated for 2 h at room temperature.
Washed blocked wells twice with PBS (V=200 µl)

Measuring:

Measured the emission from top, Measured the emission from bottom

Measured the plate from 05.08.2016 again with only one blank:

fig.1: The Average over replicates of fluorescence in RFU. Measured the fluorescence of coated GFP (20 µg/ml) and the auto fluorescence of empty wells as a negative control, by measuring from top with the plate reader.

fig.2: The Average over replicates of fluorescence in RFU. Measured the fluorescence of coated GFP (20 µg/ml) and the auto fluorescence of empty wells as a negative control, by measuring from bottom with the Plate reader.

Prepared coating solutions with several concentrations:

20 µg/ml
28.7 µg/ml
41.8 µg/ml
35.6 µg/ml

07.08.2016

Coated MED. Binding Greiner Plate:

Coated the wells with 50 µl from prepared coating solution:

fig.3: The Average over replicates of fluorescence in RFU. Measured the fluorescence of coated GFP (20 µg/ml, 28.7 µg/ml, 41.8 µg/ml and 35.6 µg/ml) and the auto fluorescence of empty wells as a negative control, by measuring from top with the Plate reader.

fig.4: The Average over replicates of fluorescence in RFU. Measured the fluorescence of coated GFP (20 µg/ml, 28.7 µg/ml, 41.8 µg/ml and 35.6 µg/ml), the auto fluorescence of empty wells as a negative control and the fluorescence of GFP (20 µg/ml, 28.7 µg/ml, 41.8 µg/ml and 35.6 µg/ml) in solution as positive control, by measuring from top with the plate reader.

08.08.2016

Coated

Used Greiner MID bind plate
Spores: WT, B53, B54; used 50 µl/well, spores were purified by washing with PBS, to remove the medium.
used GFP coating concentrations of: 40 µg/ml, 45 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 90 µg/ml; used 50 µl/well
m.Cherry: 20 µg/ml, 40 µg/ml, 80 µg/ml

coated over night at 4°C in the fridge.

09.08.2016

Blocked

Washed plate from previous day twice with 200 µl PBS
Incubated for 2 h at room temperature
Blocked with 200 µl 5% nonfat dry milk in PBS
Incubated for 2 h at room temperature

Measured

Measured the fluorescence at two different focal heights:
22mm:

fig.5: Average of fluorescence over replicates of samples (B53_coated 18 mill., B53_solution 18 mill., B53_coated 9 mill., B53_solution 9 mill., B53_coated 1.8 mill., B53_solution 1.8 mill., B54_cated 18 mill., B54_solution 18 mill., B54_coated 9 mill., B54_solution 9 mill., B54_coated 1.8 mill., B54_solution 1.8 mill., GFP_coated 20 µg/ml, GFP_coated 35.6 µg/ml, GFP_coated 40 µg/ml, GFP_coated 45 µg/ml, GFP_coated 60 µg/ml, GFP_coated 70 µg/ml, GFP_coated 80 µg/ml, GFP_coated 90 µg/ml, m.Cherry_coated 20 µg/ml, m.Cherry_coated 40 µg/ml, m.Cherry_coated 80 µg/ml, WT_coated 18 mill.) and an empty well as negative control, at a focal height of 22 mm.

2 mm:

fig.6: Average of fluorescence over replicates of samples (B53_coated 18 mill., B53_solution 18 mill., B53_coated 9 mill., B53_solution 9 mill., B53_coated 1.8 mill., B53_solution 1.8 mill., B54_cated 18 mill., B54_solution 18 mill., B54_coated 9 mill., B54_solution 9 mill., B54_coated 1.8 mill., B54_solution 1.8 mill., GFP_coated 20 µg/ml, GFP_coated 35.6 µg/ml, GFP_coated 40 µg/ml, GFP_coated 45 µg/ml, GFP_coated 60 µg/ml, GFP_coated 70 µg/ml, GFP_coated 80 µg/ml, GFP_coated 90 µg/ml, m.Cherry_coated 20 µg/ml, m.Cherry_coated 40 µg/ml, m.Cherry_coated 80 µg/ml, WT_coated 18 mill.) and an empty well as negative control, at a focal height of 2 mm.

13.08.2016

Dried in PBS

Scheme:

fig.7: Pipetting scheme for the measuring of fluorescence at the surface with several concentrations of GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml) and two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54). The shaded wells show wells with PBS dried, where the solution of GFP/spores was dried at 37°C in the incubator.

took from B54 and B53 aliquot 108 µl (108 mill. Spores) and 54µl (54 mill. spores)
washed them by spinning down at 13000rpm for 13min and resuspended the pellet in PBS, repeated three times
resuspended final pellet in 300µl PBS -pipetted 50 µl solution into the shaded wells (scheme)
incubated at 37°C for 5,5 h

Measured

Measured the plate with different focal heights and gain values:

Focal height 22 mm, gain 1740
Focal height self-adjusted at 3.6 mm, gain 1740
Focal height 3.6 mm, gain 2960

Result

Dry

fig.8: Average of relative fluorescence over replicates of dried in PBS, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. Used setting: focal height of 22 mm and gain value of 1740.

fig.9: Average of relative fluorescence over replicates of dried in PBS, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. Used setting: focal height of 3.6 mm and gain value of 1740.

fig.10: Average of relative fluorescence over replicates of dried in PBS, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. Used setting: focal height of 3.6 mm and gain value of 2960.

Wet:

fig.11: Average of relative fluorescence over replicates with PBS on top GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 22 mm and gain value of 1740.

fig.12: Average of relative fluorescence over replicates with PBS on top GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6 mm and gain value of 1740.

fig.13: Average of relative fluorescence over replicates with PBS on top GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. Used setting: focal height of 3.6 mm and gain value of 2960.

14.08.2016 – 15.08.2016

Dried in H2O

scheme:

fig.14: Pipette scheme for the measuring of fluorescence at the surface by several concentrations of GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml) and two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54). The shaded wells shown dried wells with H2O, were the solution of GFP/spores was dried by 37°C in the incubator.

Preparation:

Prepared dilution series of GFP and spores in H2O as in the scheme
Pipetted 100 µl per well and incubated it at 37°C for 12 h 40 min.

Measured:

Measured the plate with three different settings:
* Focal height 22 mm, gain 1740 * Focal height 3.6 mm, gain 1740 * Focal height 3.6 mm, gain 2960

Result:

Focal height 22 mm, gain 1740:

fig.15: Average of relative fluorescence over replicates of dried in H2O, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. Used setting: focal height of 22 mm and gain value of 1740.

Focal height 3.6 mm, gain 1740:

fig.16: Average of relative fluorescence over replicates of dried in H2O, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6 mm and gain value of 1740.

Focal height 3.6 mm, gain 2960:

fig.17: Average of relative fluorescence over replicates of dried in H2O, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. Used setting: focal height of 3.6 mm and gain value of 2960.

16.08.2016 – 15. 08.2016

Coated

Coated free wells on the plate where GFP dried in with PBS, used wells D5 – D12, H5 – H12 and D1 as blank
Coated concentrations of: 20 µg/ml (D5 – D7), 15 µg/ml (D8 – D10), 10 µg/ml (D11, D12 and H5), 5 µg/ml (H6 – H8)
Coated with 50 µl
Incubated for 2 h at room temperature
Washed wells twice with 200 µl PBS
Blocked with 200 µl 5% nonfat dry milk in PBS
Incubated for 2 h at room temperature
Washed the wells twice with 200 µl PBS

Measured

Measured the emission of the fluorescence with several settings:
Focal height 22 mm, gain 1740
Focal height 3.6 mm, gain 174
Focal height 3.6 mm, gain 2960

Result

Focal height 22 mm, gain 1740:

fig.18: Measured the relative fluorescence over replicates of coated GFP (20 µg/ml, 15 µg/ml, 10 µg/ml, 5 µg/ml) and empty wells as negative control, at focal height 22 mm with a gain value of 1740.

Focal height 3.6 mm, gain 1740:

fig.19: Measured the relative fluorescence over replicates of coated GFP (20 µg/ml, 15 µg/ml, 10 µg/ml, 5 µg/ml) and empty wells as negative control, at focal height 3.6 mm with a gain value of 1740.

fig.20: Measured the relative fluorescence over replicates of coated GFP (20 µg/ml, 15 µg/ml, 10 µg/ml, 5 µg/ml) and empty wells as negative control, at focal height 3.6 mm with a gain value of 2960.

19.08.2016

Plate coated and washed

Coated

Used Greiner HIGH bind plate
Coating concentration of GFP 20 µg/ml
Coating volume 50 µl
Incubated for 4 h at room temperature

Blocked

Washed coated wells twice with 200 µl PBS
Blocked with 200 µl 5% nonfat dry milk in PBS
Incubated for 2 h at room temperature
Washed wells twice with 200 µl PBS

Measured

Measured with a focal height of 3.6 mm and a gain value of 1740

Result

fig.20: Average of relative fluorescence units over replicates of coated GFP (20 µg/ml) and empty wells as negative control.

Washing – Assay 01

scheme:

fig.21: washed coated GFP with different detergents (SDS, Tx100, Tween20, Shampoo1 (Syoss OLEO 21) and Shampoo 2 (Herbal Essences Verwöhnende Feuchtigkeit), one group was washed with 2% SDS as positive control, empty was washed with PBS and wasn’t coated with GFP, the positive control was washed with PBS.

Concentration for each detergent:
SDS: 2%; 0,1%; 0,01%; 0,001%; 0,0001%
Triton: 0,1%; 0,01%; 0,001%; 0,0001%
Tween: 0,1%; 0,01%; 0,001%; 0,0001%
Shampoo 1: 0,1%; 0,01%; 0,001%; 0,0001%
Shampoo 2: 0,1%; 0,01%; 0,001%; 0,0001%

Washed the plate after the scheme with 200 µl detergent and shook for 2 min, pipetted out.
Measured the fluorescence of GFP
Washed with PBS
Measured the fluorescence of GFP
Washed again with 200 µl detergent and shook for 2 min, pipetted out
Measured the fluorescence of GFP
Washed with PBS
Measured the fluorescence of GFP

Result

21.08.2016

Washing – Assay 02

Prepared different concentrations of detergents:

SDS: 2%; 1,5%; 1%; 0,5%; 0,1%
Triton: 1,5%; 1%; 0,5%; 0,1%
Tween: 1,5%; 1%; 0,5%; 0,1%
Shampoo 1: 1,5%; 1%; 0,5%; 0,1%
Shampoo 2: 1,5%; 1%; 0,5%; 0,1%

For washing used plate and scheme from 19.06.2016 with different concentrations.
washed the coated wells with the detergents, pipetted 200 µl detergent and shook for 2 min, pipetted out.
Measured the fluorescence emission after washing with detergent.
Measured the fluorescence emission after washing with PBS.

Measured

after washing with detergents
washed with PBS afterwards

Results

fig.24: RFU difference between last measurement of the plate (washed again with PBS) and washed with detergents: SDS (2%; 1,5%; 1%; 0,5%; 0,1%), Triton: 1,5%; 1%; 0,5%; 0,1%), Tween (1,5%; 1%; 0,5%; 0,1%), Shampoo 1 (1,5%; 1%; 0,5%; 0,1%), Shampoo 2 (1,5%; 1%; 0,5%; 0,1%), SDS 2% was used as positive control. Used a focal height of 3.6 mm and a gain value of 1740.

23.08.2016

GST-Assay 01

Preparation

Prepared dilution of GST stock (5 Units/ml):

1 U/ml
0.75 U/ml
0.5 U/ml
0.1 U/ml
0.02 U/ml
0.001 U/ml

Volume master mix 1.5 ml: (15 µlCDNB [100 mM] + 15 µl GSH [100 mM] + 1470 µl PBS).

Plate scheme:

fig.25: scheme for measuring the activity of different GST concentrations (1 U/ml, 0.75 U/ml, 0.5 U/ml, 0.1 U/ml, 0.02 U/ml, 0.001 U/ml), Empty (PBS and master mix), blank (PBS and master mix).

Measured

start the reaction with 30 µl GST as in the scheme (Abb.27), in blank and empty (control) 30 µl PBS was added.
Setrtings:protocol_gst_run_1_.xlsx

Result

All data and graphs were collected in an Excel file: freiburg_2016_gst_run_1.xlsx

GST-Assay 02

Preparation

prepared samples:

0.3 U (2x 0.15 U) 0.15 U (90 µl of 5 U/ml stock)
0.25 U (2x 0.125 U) 0.125 U (25 µl of 5 U/ml stock + 5 µl PBS)
0.2 U (2x 0.1 U) 0.1 U (20 µl of 5 U/ml stock + 10 µl PBS)
0.15 U (2x 0.075 U) 0.075 U (15 µl of 5 U/ml stock + 15 µl PBS)
0.1 U (2x 0.05 U) 0.05 U (10 µl of 5 U/ml stock + 20 µl PBS)
0.05 U (2x 0.025 U) 0.025 U (5 µl of 5 U/ml stock + 25 µl PBS)
0.02 U (2x 0.01 U) 0.01 U (2 µl of 5 U/ml stock + 28 µl PBS)

volume of each sample contains 30 µl.
Prepared mester mix (1470 µl PBS + 15 µl GSH [100 mM] + 15 µl CDNB [100 mM]).
Pipetted GST samples like in the scheme (fig.26)

Scheme:

fig.26: pipetting scheme of GST amount (0.15 U, 0.125 U, 0.1 U, 0.075 U, 0.05 U, 0.025 U, 0.01 U), white=empty (master mix and PBS) and black=blank (master mix and PBS).

Measured

Started the reaction with 90µl master mix in each well
Put it immediately in the plate reader
Used measurement settings:protocol_gst_run_2_.xlsx

Results

All data and graphs were collected in an Excel file:freiburg_2016_18.09.16_gst-assay_02_.xlsx

GST – Assay 03

Repeated the assay of GST – Assay 02
Pipetting scheme as in GST – Assay 02 (fig.26)

Measured

Start the reaction with 90µl assay cocktail in each well
Used settings as shown in the reader protocol: protocol_gst_run_3.xlsx

Results

All graphs were collected in an excel file: freiburg_2016_gst_run3.xlsx

GST – Assay 04

Repeat the assay depending on the results of GST – Assay 03 (Freiburg 2016_gst_run3.xlsx). increased the measuring time up to 10min and pipetted on ice.
Pipetted after scheme shown in fig.26.

Measured

Start the reaction with 90µl assay cocktail in each well
Put it immediately in the plate reader
Used settings as shown in the reader protocol: protocol_gst_run_4.xlsx

Results

All graphs were collected in an excel file: freiburg_2016_gst_run4.xlsx

29.08.2016

Detergent screening

Scheme:

fig.27: scheme of detergent screening for SDS, Tx100, Tween20, Shampoo1 and Shampoo2, as positive control was used a solution of 2% SDS, for negative control was used a wash solution of PBS without detergents.

Coated

Prepared GFP coating solution (20µg/ml)
Coated the plate after the scheme (fig.27) with coating volume of 100µl
Incubated for 2h at room temperature
Washed the wells twice with 200µl PBS
Blocked wells with 200µl 5% nonfat dry milk in PBS
Incubated for 2h at room temperature
Washed wells twice with 200µl PBS

30.08.2016

Preparation of detergents for screening: Prepared stock solutions of 20% and 40%
- Prepared samples of 1ml for each the three cycles (except Block three there were only two cycles) in each block:

Block 1: 2, 4, 6, 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.25, 1, 0.5 and 0.1% solutions
Block 2: 12, 14, 16,1 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.75, 1, 0.5 and 0.1% solutions
Block 3: 32, 34,3 6, 38% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 1.25, 1, 0.5 and 0.1% solutions

Washing

Block 1:

Washed with concentrations of 2, 4, 6, 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.25, 1, 0.5 and 0.1%, for negative control used PBS and for positive control 2% SDS. Wash solution volume took 200µl.
washed with PBS twice afterwards
pipetted out the whole volume which was left in the wells
measured the fluorescence
repeated it for a total of 3 cycle

Block 2:

Washed with concentrations of 12,14, 16, 18% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.75, 1, 0.5 and 0.1%, for negative control used PBS and for positive control 2% SDS. Wash solution volume took 200µl.
washed with PBS twice afterwards
pipetted out the whole volume which was left in the wells
measured the fluorescence
repeated it for a total of 3 cycle

Block 3:

Washed with concentrations of 32,34, 36, 38% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 1.25, 1, 0.5 and 0.1%, for negative control used PBS and for positive control 2% SDS. Wash solution volume took 200µl.
washed with PBS twice afterwards
pipetted out the whole volume which was left in the wells
measured the fluorescence
repeated it

Result

Data and figures were collected in an excel file:freiburg_2016_30.08.16_detergengt_screening.xlsx

Fig.28: wash efficiency of detergents in an overview.

02.09.2016

Washing – Assay 03

Plate name W5
coated the plate with GFP after coating protocol, used scheme from fig.29

scheme:

Fig.29: washing scheme with several concentrations of detergents: SDS: 1.25, 1, 0.75, 0.25%; Tween20: 22, 24, 26, 28, 32, 24, 26, 28%; SH1: 12, 14, 16 ,18, 22, 24, 26, 28%. For neg. contr. used 2% SDS, pos. contr. was washed with PBS without detergent.

stock solution 40%

measured

used setting for measuring the fluorescence form coated GFP and washed plate as shown in the protocol:

Result

Detailed figures collected in an excel file:

Fig.30: overview of the wash efficiency of detergents: SDS: 1.25, 1, 0.75, 0.25%; Tween20: 22, 24, 26, 28, 32, 24, 26, 28%; SH1: 12, 14, 16 ,18, 22, 24, 26, 28%. For neg. contr. used 2% SDS, pos. contr. was washed with PBS without detergent.

GST – Assay 05

repeated the assay depending on the results of GST – Assay 04 (excel file: Freiburg 2016gst_run4.xlsx.

scheme:

fig.26: pipetting scheme of GST-amount (0.15U, 0.125U, 0.1U, 0.075U, 0.05U, 0.025U, 0.01U), white empty (Assay cocktail and PBS) in black Blank (Assay cocktail and PBS).

prepared the samples on ice
incubated the plate before started in the fridge for 5min.
prepared samples after the scheme shown in fig.26

Measured

started the reaction with 90µl assay cocktail in each well
put it immediately in the plate reader
used settings as shown in the reader protocol: protocol_gst_run_5.xlsx

Result

All data and graphs were collected in an excel file: freiburg_2016_gst_run5.xlsx

03.09.2016

GST – Assay 06

Preparation as like in GST – Assay 05. Took a little more time to start the measuring after mixing enzyme and assay cocktail
Used pipetting scheme as shown in fig.26.

Measuring

Used setting as shown in the reader protocol: protocol_gst_run_6.xlsx

Result

All data and graphs were collected in an excel file: freiburg_2016_gst_run6.xlsx

GST – Assay 07

Repeated like as in assay 05, but all was pipetted on ice. Used pipetting scheme as shown in fig.26.

Measured

Start the reaction with 90µl assay cocktail from low amount of GST concentration to high, for reducing the time delay the assay cocktail was piped in directly next to the plate reader

Result

All data and Graphs were collected in an excel file: freibur_2016_gst_run7.xlsx

GST – Assay 08

Preparation

Preparations like as in the other Assays before (GST – Assay 07) after pipetted and incubate for 5min in the fridge put the plate on a shaker.

Measuring

started the reaction with 90 µl Assay cocktail while shaking at 300rpm and temperature (temperature of the block) of 12°C.
Put it then directly into the reader.
protocol: protocol_gst_run_8.xlsx

Result

graphs were collected in an excel file: freiburg_2016_gst_run8.xlsx

GST-Assay 09

Preparation

* as in GST-Assay 8 * increased measuring time up to 20min in the measuring settings

Measuring

Started the reaction like in GST-Assay 8, used settings as shown in the protocol: protocol_gst_run_9.xlsx

Result

All data and graphs were in an excel file: freiburg_2016_gst_run9.xlsx

12.09.2016

GST – Assay 10

Perpetrated

samples of GST:

0.15U (90µl of 5U/ml stock)
0.075U (15µl of 5U/ml stock + 75µl PBS)
0.025U (5µl of 5U/ml stock + 85µl PBS)
0,005U (3µl of 5U/ml stock + 87µl PBS)

Assay cocktail: 15µl CDNB + 15µl GSH + 1470µl PBS.
Used 30µl enzyme solution and 90µl Assay cocktail per well, for Blank and Empty used 30µl PBS and Assay cocktail.

Scheme:

fig.31: Pipetting scheme for GST – Assay wit 0.15, 0.075, 0.025 and 0.005 Units of GST and Assay cocktail, for wells empty and blank used instead of GST solutions PBS and Assay cocktail.

Assay cocktail wasn’t enough for all wells, concentration of 0.15U without assay cocktail.

Measured

Used setting as shown in reader protocol:

Result

Data and Graphs were collected in an excel file:

GST – Assay 11

Repeated GST – Assay 10 with more assay cocktail, scheme fig.31
1,7ml: 17µl CDNB + 17µl GSH + 1666µl PBS

Measured

Used setting as shown in reader protocol:

Result

Data and Graphs were collected in an excel file:

GST – Assay 12

Repeated measurement of GST – Assay 11 with different measurement settings.
Used scheme the same scheme fig.31

Measured

Used setting as shown in reader protocol:

Result

Data and Graphs were collected in an excel file:

14.09.2016

Shampoo: washing with nanobody

Prepared

Coated plate after the scheme
Prepared nanobody solution: Concentration nanobody stock solution 0.48mg/ml, set up dilution of 1.8ng/µl
Scheme:

fig.32: washing scheme for washing with nanobody and detergents. As control were used: GFP washed with PBS (negative control 1), washed with nanobody in PBS (negative control 2), 2%SDS (positive control). As reference were empty wells not coated and unwashed, same as the blank. Used detergent samples: SDS: 1, 0.75, 0.5, 0.25% solutions with nanobody, Tween20: 22, 24, 26,28% solutions with nanobody.

wash – assay

Measured the fluorescence of coated GFP
Washed with 1µl nanobody and 200µl concentrations of detergents: SDS: 1.5, 1, 0.5, 0.25%, Tween20: 22, 24, 26, 28%, SH1: 12, 14, 16, 18%
Incubated it for 5min
Shook it for 5min at 300rpm
washed with PBS twice afterwards
pipetted out the whole volume which was left in the wells
measured the fluorescence, used same settings as measured the fluorescence of coated GFP:

Result

Detailed column charts were collected in an excel file: washing_with_nanobody_eff..xlsx

fig.33: overview of the efficiency of washing with nanobody in combination with detergents. SDS: 1, 0.75, 0.5, 0.25% solutions with nanobody, Tween20: 22, 24, 26,28% solutions with nanobody. Controls GFP washed with PBS (negative control 1), washed with nanobody in PBS (negative control 2), 2%SDS (positive control).

14.09.2016

GST – Assay 13

Scheme:

fig.34: pipetting scheme of GST – Assay, used GST amount of: 0.0001, 0.00001, 0.000001, 0.0000001 Units, for empty and blank was used PBS instead of GST.

Measured

Measurement settings as in the reader protocol:14.09.16_gst-assay_settings.xlsx

Result

All data and graphs were collected in an excel file: freiburg_2016_14.09.16_gst-assay_-1.xlsx

16.09.2016

Shampoo

Platename: WN2
Scheme:
Fig.35: washing scheme for washing with nanobody and detergents. As control were used: coated GFP not washed (negative control 1), GFP washed with PBS (negative control 2), washed with nanobody in PBS (negative control 3), 2%SDS (positive control). As reference were empty wells not coated and unwashed, same as the blank. Used detergent samples: SDS: 1, 0.75, 0.5, 0.25% solutions with nanobody, Tween20: 22, 24, 26,28% solutions with nanobody.

Prepared

coated plate with GFP (coating concentration 20µg/ml)
dilute 1µl nanobody stock solution (0.48mg/ml) in prepared samples 1ml detergents: SDS: 1, 0.75, 0.5, 0.25, Tween20: 22, 24, 26, 28%, SH1: 12, 14, 16, 18%

Wash-assay

Measured the fluorescence of coated GFP
Washed with 200µl detergents: SDS: 1.5, 1, 0.5, 0.25% (Row A and B with nanobody, row C and D without nanobody), Tween20: 22, 24, 26, 28% (Row A and B with nanobody, row C and D without nanobody), SH1: 12, 14, 16, 18% (Row E and F with nanobody, row G and H without nanobody)
Incubated it for 5min
Shook it for 5min at 300rpm
washed with PBS twice afterwards
pipetted out the whole volume which was left in the wells
measured the fluorescence
used same settings as measured the fluorescence of coated GFP:

Result

Detailed column charts were collected in an excel file:freiburg_2016_16.09.16_washing_nanobody_02_gfp_master.xlsx

Fig.36: Overview of the wash efficiency between with and without nanobody for: SDS, SDS + nanobody, Tween20, Twenn20 + nanobody, SH1, SH1 + nanobody.

17.09.2016

GST – Assay 14

Repeat the GST – Assay 13 with the same scheme (fig34).
Concentration of samples:

0,0001U (3µl 0,1U/ml stock + 87µl PBS)
0,00001U (3µl 0,01U/ml stock + 87 PBS)
0,000001U (3µl 0,001U/ml stock + 87 PBS)
0,0000001U (3µl 0,0001U/ml stock + 87 PBS)

Assay cocktail: 15µl CDNB + 15µl GSH + 1470µl PBS.
Pipetted the GST after the scheme (30µl per well), used for empty wells and blank instead of GST solution PBS.

Measured

Start the reaction with 90µ assay cocktail from low concentration to high concentration, pipetted directly next to the plate reader to recuse the time delay.
Measured the absorbents with settings as described in the measurement protocol: 17.09.16_gst-assay_01_settings.xlsx

GST – Assay 15

Repeated the GST – Assay 14
settings: 17.09.16_gst-assay_02_settings.xlsx

GST – Assay 16

Changed setting for the measurement: shook in plate reader before each cycle at 500rpm 1s.

Scheme:

fig.37: GST – Assay pipetting scheme for GST concentrations of 0.005 and 0.0000001 Units. Empty as negative control with PBS instead of GST, blank as the same conditions like empty.

prepared

samples of 0.005 and 0.0000001U
assay – cocktail (14µl CDNB + 14µl GSH + 1372µl PBS)
pipetted GST-samples after the scheme and PBS for the controls (30µl)

Measurement

start the reaction with 170µl Assay cocktail. Used new setting as in the reader protocol described: 17.09.16_gst-assay_03_settings.xlsx

Result

All data and graphs of GST - Assay 14 - 16 were collected in an excel file: freiburg_2016_17.09.16_gst-assay_master.xlsx

18.09.2016

GST – Assay 17

Doubled the amount of substrate
Scheme:

Fig.38: Pipetting scheme for GST – Assay with 0.1, 0.025, 0.005 and 0.001 Units (U), empty wells and blank measured with PBS instead of GST as control.

Prepared

GST – samples:

0.1U (60µl of 5U/ml stock + 30µl PBS)
0.025U (5µl of 5U/ml stock + 87µl PBS)
0.005U (3µl of 5U/ml stock + 85µl PBS)
0,001U (3µl of 1U/ml stock + 87µl PBS)

Assay cocktail: 30µl CDNB + 30µl GSH + 1440µl PBS.
Pipet GST – samples and PBS after the scheme (fig.38).

Measured

Start the reaction with 90µl assay cocktail
Start measuring with settings which are described in the protocol: 18.09.16_gst-assay_01_settings.xlsx

Result

Data and graphs were collected in an excel file:18.09.16_gst-assay_01.xlsx

GST – Assay 18

Scheme:

fig.39: Pipetting scheme for GST – Assay with 0.1, 0.05 Units (U), empty wells and blank measured with PBS instead of GST as control, grey wells are not used.

Increased the measuring time up to 33 minutes.

Prepared

GST – Samples:

0,1U
0,05U (30µl of 5U/ml stock + 60µl PBS)
0,025U

Assay cocktail

Measurement

Start reactions with 90µl assay cocktail Used setting as decried in the measurement protocol of the reader: 18.09.16_gst-assay_02_settings.xlsx

Result

Data and graphs were collected in an excel file: freiburg_2016_18.09.16_gst-assay_02_-1.xlsx

19.09.2016

GST – Assay 19

Scheme:

fig.40: Pipetting scheme for GST – Assay with 0.1, 0.05 Units (U), empty wells and blank measured with PBS instead of GST as control, grey wells are not used.

Prepared

GST concentrations:

0,1U (80µl 5U/ml stock + 40µl PBS)
0,05U (40µl 5U/ml stock + 80µl PBS)

Assay cocktail: 18µl CDNB + 18µl GSH + 1164µl PBS
Changed shook time before the first cycle in the setting to 200rpm for 1s
Piped the samples after the scheme in fig.42

Measurement

Start the reactions with 90µl PBS and start measurement with the setting as described in the plate reader protocol: 19.09.16_gst-assay_01_settings.xlsx

GST – Assay 20

Repeat the GST – Assay 19 in addition with one well of enzyme without assay cocktail
Scheme:

Fig.41: Pipetting scheme for GST – Assay with 0.1, 0.05 Units (U), empty wells and blank measured with PBS instead of GST as control, En well which contains only enzyme without assay cocktail, grey wells are not used.

Prepared

GST concentrations:

0,1U (80µl 5U/ml stock + 40µl PBS)
0,05U (40µl 5U/ml stock + 80µl PBS)

Assay cocktail: 18µl CDNB + 18µl GSH + 1164µl PBS
For En (fig.41) used 30µl of 1U GST solution
Piped the samples after the scheme in fig.41

Measurement

Start the reactions with 90µl PBS and start measuremen.

Result

All data and graphs of GST - Assay 19 and 20 were collected in an excel file: freiburg_2016_19.09.16_gst-assay_master-1.xlsx

23.09.2016

GST – Assay 21

Scheme:

Fig.42: Pipetting scheme for GST – Assay with 0.1, empty wells and blank measured with PBS instead of GST as control, grey wells are not used.

Prepared

Sample of 0.1U and assay cocktail: 70µl CDNB + 70µl GSH + 560µl PBS, increased amount of substrates.

Measurement

Start the reaction with 90µ assay cocktail.
Start the measurement wit settings as before, which are described also in the reader protocol: 23.09.16_gst-assay_01_settings.xlsx

GST – Assay 22

Reduced amount of substrates from 70µl to 50µl and increased volume from 90µ to 110µl used blank of GST – Assay 21 (scheme fig.42)
Scheme:

Fig.43: Pipetting scheme for GST – Assay 22. 0.1Units GST for sample wells and as referents Empty which contains PBS and assay cocktail.

Prepared

Prepared 0.1U samples of GST and assay cocktail: 50µl CDNB + 50µl GSH + 900µl PBS
pipetted 30µl GST and PBS after the scheme (fig.43)

Measurement

Start the reactions with 110µl assay cocktail
started measurement with setting which are described in the plate reader protocol:23.09.16_gst-assay_02_settings.xlsx

Result: All data and graphs of GST - Assay 22 and 23 were collected in an excel file: freiburg_23.09.16_gst-assay_master-1.xlsx

lab book, labor

26.09.2016

GST assay

with E.coli cell lysate
neg. control: DH5α
pos. control: 0,05U GST
constructs 131, 134 & 138 (concentrations: 0,05 0,075 0,1 [µg/µl] protein per well)

Didn't work, the concentrations were too low.

29.09.2016

GST assay

concentrations: 0,1U 0,08U 0,06U 0,04U
0,08U: 48µl 5U/ml stock + 60µl PBS
0,06U: 36µl stock + 72µl PBS
0,04U: 24µl stock + 96µl PBS
1,7ml assaycocktail: 85µl CDNB + 85µl GSH + 1530µl PBS
40µl enzyme solution + 100µl assaycocktail per well

Didn't work, maybe pipetting error.

30.09.2016

GST assay

E.coli lysate

construct 134
15µl per well: 30µl lysate + 150µl PBS → 69µg proteins per well
30µl per well: 60µl lysate + 120µl PBS → 138µg proteins per well
60µl per well: 120µl lysate + 60µl PBS → 276µg proteins per well
90µl per well: 180µl lysate → 414µg proteins per well
60µl + GST per well: 120µl lysate + 0,05U (20µl 5U/ml stock + 40µl PBS)

DH5α
30µl per well: 60µl lysate + 120µl PBS → 57µg proteins per well

0,1U
40µl 5U/ml stock + 140µl PBS

1,5ml assaycocktail: 75µl CDNB + 75µl GSH + 1350µl PBS

90µl sample + 90µl assaycocktail per well

30.09.16_gst-assay_01_settings.xlsx

GST assay

E.coli lysate
constructs 131 & 138

131
28µl → 148,4µg proteins
52µl → 275,6µg proteins
75µl → 397,5µg proteins

138
25µl → 147,5µg proteins
47µl → 277,3µg proteins
68µl → 401,2µg proteins

DH5α
79µl → 150,1µg proteins

DH5α+
79µl + 0,05U (10µl stock + 1µl PBS)

0,05U

1,9ml assaycocktail: 95µl CDNB + 95µl GSH + 1710µl PBS

90µl sample + 90µl assaycocktail

Results

30.09.16_gst-assay_master.xlsx

02.10.2016

GST assay

GST-spores
constructs: Z+GST & WT+GST
concentrations: 50 million, 25 million, 10 million, 5 million spores
stock 100 million spores in 500µl
50 million: 250µl stock per well
25 million: 125µl stock per well + 125µl PBS
10 million: 50µl stock per well + 200µl PBS
5 million: 25µl stock per well + 225µl PBS

WT as control: 250µl per well

0,05U GST

1,4ml assaycocktail: 70µl CDNB + 70µl GSH + 1260µl PBS

250µl sample + 90µl assaycocktail per well

done again with the CotZ + GST construct and the CotZ construct purified

Results

The spores don't show a difference between GST-construct spores and WT spores or show no activity at all.

03.10.16

GST assay

measured E.coli lysates again with about 150µg, 275µg and 400µg of proteins

Results

The GST-spores showed no difference in activity compared to the wild type.

GST assay

measured the WT + GST spores purified
concentrations: 50million, 25million, 10million, 5million spores
controls: pos. 0,05U GST & neg. untransformed WT spores

Results

The spores showed no activity.

04.10.16

GST-assay

measured assaycocktail with freshly prepared GSH in ethanol and freshly prepared GSH in demineralized water
400µl assaycocktail: 20µl CDNB + 20µl GSH + 360µl PBS

Results

The GSH dissolved in EtOH is a little bit more stable.

05.10.16

GST-assay

measured 0,1U; 0,08U; 0,06U; 0,04U
3ml assaycocktail: 150µl CDNB + 150µl GSH + 2700µl PBS

Results

There's a measurable difference between the Units, but it's still a slight curve.

05.10.16_gst-assay_0_1u_-_0_04u.xlsx

06.10.16

GST-assay

measured E.coli lysates
DH5α: 6,8µg/µl proteins
138: 6,8µg/µl
134: 6,3µg/µl

concentrations: ~400µg; ~500µg; ~600µg per well

controls: 0,05U, Empty(PBS+assaycocktail)

06.10.16_gst-assay_lysates_settings.xlsx
06.10.16_10.10.16_gst-assay_lysates_master.xlsx

GST-assay

measured CotZ-GST knock-out spores and WT spores
50million - 5 million spores
washed unpurified spores 3 times with PBS

controls:
pos. 0,05U GST (4*10µl 5U/ml stock + 960µl PBS)
neg. empty (250µl PBS + 90µl assaycocktail)
untrans. WT (50-5million)

3ml assaycocktail: 150µl CDNB + 150µl GSH + 2700µl PBS

250µl sample + 90µl assaycocktail