Difference between revisions of "Team:Freiburg/NotebookDelivery"

1. 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.
   
2. Dilute stock solution of GFP (1350µg/ml) in coating buffer for coating concentration of 20 µg/ml.
   
3. Coated 93 wells with 50µl coating solution
   
4. Incubated for 2 h at room temperature
   
5. Washed the wells twice with PBS (V=200 µl)
   
6. Blocked it with 5% nonfat dry milk in PBS (2.5 g in 50 ml PBS), blocked coated wells with 200 µl solution.
   
7. Incubated for 2 h at room temperature.
   
8. Washed blocked wells twice with PBS (V=200 µl)
   

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

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.

Coated

1. Used Greiner MID bind plate
2. Spores: WT, B53, B54; used 50 µl/well, spores were purified by washing with PBS, to remove the medium.
3. 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
4. m.Cherry: 20 µg/ml, 40 µg/ml, 80 µg/ml

coated over night at 4°C in the fridge.

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

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.

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.

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

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

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.

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.

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.

• 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 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

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.

• 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 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

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.

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

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

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

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

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%

1. Washed the plate after the scheme with 200 µl detergent and shook for 2 min, pipetted out.
2. Measured the fluorescence of GFP
3. Washed with PBS
4. Measured the fluorescence of GFP
5. Washed again with 200 µl detergent and shook for 2 min, pipetted out
6. Measured the fluorescence of GFP
7. Washed with PBS
8. Measured the fluorescence of GFP

Setting:gfg_highbind_deterg_settings.xlsx

Setting:gfp_highbind_deterg_pbs_settings.xlsx

Setting:gfp_highbind_againdet-settings.xlsx

Setting:gfp_highbind_againdet_pbs-settings.xlsx

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.

after washing with detergents
washed with PBS afterwards

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.

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).

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

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

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).

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

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

Repeated the assay of GST – assay 01
Pipetting scheme as in GST – assay 01 (fig.26)

• Start the reaction with 90 µl master mix in each well
• Used settings as shown in the reader protocol: protocol_gst_run_3.xlsx

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

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

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

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

Scheme:

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

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

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

• block 1: 2, 4, 6, 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Shampoo2 (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 Shampoo2 (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 Shampoo2 (SH2); for SDS used concentrations 1.5, 1.25, 1, 0.5 and 0.1% solutions

Block 1:

1. Washed with solutions with concentrations of 2, 4, 6, 8% 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 200 µl.
2. washed with PBS twice afterwards
3. pipetted out the whole volume which was left in the wells
4. measured the fluorescence
5. repeated it for a total of 3 cycles

block 2:

1. Washed with solutions with concentrations of 12,14, 16, 18% of Tx100, Tween20, Shampoo1 (SH1) and Shampoo2 (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 200 µl.
2. washed with PBS twice afterwards
3. pipetted out the whole volume which was left in the wells
4. measured the fluorescence
5. repeated it for a total of 3 cycles

block 3:

1. Washed with solutions with concentrations of 32,34, 36, 38% of Tx100, Tween20, Shampoo1 (SH1) and Shampoo2 (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 200 µl.
2. washed with PBS twice afterwards
3. pipetted out the whole volume which was left in the wells
4. measured the fluorescence
5. repeated it

Data and graphs were collected in an Excel file:freiburg_2016_30.08.16_detergengt_screening.xlsx

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

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%

12% | 300 µl stock solution + 700 µl PBS
14% | 350 µl stock solution + 650 µl PBS
16% | 400 µl stock solution + 600 µl PBS
18% | 450 µl stock solution + 550 µl PBS
22% | 550 µl stock solution + 450 µl PBS
24% | 600 µl stock solution + 400 µl PBS
26% | 650 µl stock solution + 350 µl PBS
28% | 700 µl stock solution + 300 µl PBS
32% | 800 µl stock solution + 200 µl PBS
34% | 850 µl stock solution + 150 µl PBS
36% | 900 µl stock solution + 100 µl PBS
38% | 950 µl stock solution + 50 µl PBS

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

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.

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

scheme:

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

1. prepared the samples on ice
2. incubated the plate before starting in the fridge for 5 min.
3. prepared samples after the scheme shown in fig.26

1. started the reaction with 90 µl master mix in each well
2. put it immediately in the plate reader
3. used settings as shown in the reader protocol: protocol_gst_run_5.xlsx

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

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

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

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

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

Start the reaction with 90 µl master mix from low GST concentration to high, for reducing the time delay the master mix was pipetted directly next to the plate reader

All data and graphs were collected in an Excel file: freibur_2016_gst_run7.xlsx

Preparations like in the other assays before (GST – assay 07) after pipetting and incubating for 5 min in the fridge, put the plate on a shaker.

started the reaction with 90 µl master mix while shaking at 300rpm and shaker temperature of 12°C.
Put it then into the reader.
protocol: protocol_gst_run_8.xlsx

graphs were collected in an Excel file: freiburg_2016_gst_run8.xlsx

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

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

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

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

master mix: 15 µl CDNB + 15 µl GSH + 1470 µl PBS.
Used 30 µl enzyme solution and 90 µl master mix per well, for blank and empty used 30 µl PBS and master mix.

Scheme:

fig.31: Pipetting scheme for GST – assay with 0.15, 0.075, 0.025 and 0.005 Units of GST and master mix, for wells empty and blank used instead of GST solutions PBS and master mix.

master mix wasn’t enough for all wells, concentration of 0.15 U without master mix.

Used setting as shown in reader protocol:

Data and graphs were collected in an Excel file:

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

Used setting as shown in reader protocol:

Data and graphs were collected in an Excel file:

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

Used setting as shown in reader protocol:

Data and graphs were collected in an Excel file:

Coated plate after the scheme
Prepared nanobody solution: Concentration nanobody stock solution 0.48 mg/ml, set up dilution of 1.8 ng/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 empty wells were 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.

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

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).

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.

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

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

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.

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

1. Measured the fluorescence of coated GFP
2. Washed with 200 µl 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)
3. Incubated it for 5 min
4. Shook it for 5 min at 300 rpm
5. washed with PBS twice afterwards
6. pipetted out the whole volume which was left in the wells
7. measured the fluorescence
8. used same settings as measured the fluorescence of coated GFP:
   

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 with and without nanobody for: SDS, SDS + nanobody, Tween20, Twenn20 + nanobody, SH1, SH1 + nanobody.

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

• 0,000 1U (3 µl 0,1 U/ml stock + 87 µl PBS)
• 0,00001 U (3 µl 0,01 U/ml stock + 87µl PBS)
• 0,000001 U (3 µl 0,001 U/ml stock + 87µl PBS)
• 0,0000001 U (3 µl 0,0001 U/ml stock + 87µl PBS)

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

Start the reaction with 90 µl mastr mix from low concentration to high concentration, pipetted next to the plate reader to reduce the time delay.
Measured the absorbance with settings as described in the measurement protocol: 17.09.16_gst-assay_01_settings.xlsx

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

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

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 with the same conditions like empty.

prepared

• samples of 0.005 and 0.0000001 U
• master mix (14 µl CDNB + 14 µl GSH + 1372 µl PBS)
• pipetted GST samples after the scheme and PBS for the controls (30 µl)

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

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

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.

GST samples:

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

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

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

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

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 were not used.

Increased the measuring time up to 33 minutes.

GST Samples:

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

master mix

Start reactions with 90 µl master mix. Used setting as described in the measurement protocol of the reader: 18.09.16_gst-assay_02_settings.xlsx

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

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 were not used.

GST concentrations:

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

master mix: 18 µl CDNB + 18 µl GSH + 1164 µl PBS
Changed shaking time before the first cycle in the setting to 200 rpm for 1s
Pipetted the samples after the scheme in fig.42

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

Repeat the GST – assay 19 in addition with one well of enzyme without master mix
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 were not used.

GST concentrations:

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

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

Start the reactions with 90 µl master mix and start measurement

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

Scheme:

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

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

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

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

Fig.43: Pipetting scheme for GST – assay 22. 0.1 Units GST for sample wells and as reference empty which contains PBS and master mix.

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

Start the reactions with 110 µl master mix
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

with E.coli cell lysate
neg. control: DH5α
pos. control: 0,05 U 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.

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

Didn't work, maybe pipetting error.

E.coli lysate

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

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

0,1 U
40 µl 5 U/ml stock + 140 µl PBS

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

90 µl sample + 90 µl master mix per well


30.09.16_gst-assay_01_settings.xlsx

E.coli lysate
constructs 131 & 138

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

138
25 µl → 147,8 µg proteins
47 µl → 277,3 µg proteins
68 µl → 401,2 µg proteins

DH5α
79 µl → 150,1 µg proteins

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

0,05 U

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

90 µl sample + 90 µl master mix

30.09.16_gst-assay_master.xlsx

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

WT as control: 250 µl per well

0,05 U GST

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

250 µl sample + 90 µl master mix per well

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

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

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

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

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

The spores showed no activity.

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

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

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

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

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

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

controls: 0,05 U, empty (PBS+master mix)




06.10.16_gst-assay_lysates_settings.xlsx

results see below 10.10.16

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

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

3 ml master mix: 150 µl CDNB + 150 µl GSH + 2700 µl PBS

250 µl sample + 90 µl master mix

The spores showed no activity.

with GST-CotZ(153)-, CotG-GST(156)-, GST-CotG(167)- & CgeA-GST(168)-construct spores.
50 million - 5 million spores

controls:
pos. 0,05 U GST
empty
untrans. WT

3,4 ml master mix: 170 µl CDNB + 170 µl GSH + 3060 µl PBS


09.10.16_gst-assay_settings.xlsx

see below 11.10.16

measured E. coli lysates again with only one concentration (600 µg)
controls:
pos. 0,05 U GST
empty
untrans. WT

06.10.16_10.10.16_gst-assay_lysates_master.xlsx

prepared 3 plates
tested: aGFPnano-CotZ(159WT), aGFPnano-CotZ(157WT), aGFPnano-CotG(151WT)

coated 68 wells with GFP, coating concentration GFP 20 µg/ml and coating volume 100 µl
incubated 2 h at room temerature
washed twice with 200 µl PBS
blocked with 200 µl 5% nonfat dry milk in PBS
incubated for 2 h at roomtemperatur
washed twice with PBS
measured the fluorescence of coated GFP

used 200 µl washing of solutions:

• SDS: 1%, 0.75%, 0.5%, 0.25% (with spores, nanobody and only SDS)
• Tween20: 22%, 24%, 26%, 28% (with spores, nanobody and only Tween20)
• SH1: 12%, 14%, 16%, 18% (with spores, nanobody and only SH1)
• Controls: nanobody in PBS, WT - spore in PBS, construct spore in PBS, 2% SDS & PBS

incubated wash solutions on the plate for 5 min
shook for 5 min at 300 rpm
washed twice with PBS
measured the emission of remaining GFP

bba_k2114001_agfpnano_ha_ahelix_cotz_in_wt_spores.xlsx
bba_k2114002_agfpnano_ha_g4s_cotz_in_wt_.xlsx
bba_k2114009_agfpnano_ha_ahelix_cotg_in_wt.xlsx

GST-assay

Results

11.10.16_gst-assay_spores_master.xlsx

The CotG-GST spores(156) show a difference in the slope compared to the WT spores. This difference is significant.