Group 4 - Adhesion
Members: Kevin, Christian, Vivi, Nathalie
07/04/2016
Bacillus subtilis strains used:
Strain |
B52:B. Subtilis w168 amyE::PcgeA-CotZre-gfp-B0014(CM5) |
B53:B. Subtilis w168 amyE::PcotYZ-CotZre-gfp-B0014(CM5) |
B54:B. Subtilis w168 amyE::PcotYZ-CotZin-gfp-B0014(CM5) |
B55:B. Subtilis w168 amyE::PcotV-CotZre-gfp-B0014(CM5) |
B56:B. Subtilis w168 amyE::PCotv-CotZin-gfp-B0014(CM5) |
07/05/2016
Slide preparation: GOPTS
Surface activation of glass slides by plasma generator, coated with GOPTS 3-glycidyloxypropyl trimethoxysilane, washed with acetone and then stored for up to two weeeks.
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labeled with a diamond pen
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washed with EtOH then ddH2O
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dried slides with compressed air
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activation of surface with plasma generator
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incubation with GOPTS (overnight)
07/10/2016
Buffer for anti-GFP nanobody (10 ml)
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61mg Tris
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87mg NaCl
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1ml Glycerin
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2,5 ml Imidazole(1M)
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fill up with ddH2O to 10 ml
07/12/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
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incubation with nanobody (0.5µl, 330µg/ml, overnight, 4°C)
07/13/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
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blocked with BSA(2%) (150µl/spot, 30 min)
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washed with PBS(1x)
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washed with imidazole (20mM) in PBS(1x)
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washed with PBS(1x)
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washed with ddH2O
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dried with compressed air
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incubation with B52-B56 (100µl/spot, 120 min)
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incubation with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top
Spot No. | Sample |
1 | GOPTS + nanobody + B52 |
2 | GOPTS + nanobody + B53 |
3 | GOPTS + nanobody + B54 |
4 | GOPTS + nanobody + B55 |
5 | GOPTS + nanobody + B56 |
07/14/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
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edges sealed with nail polish
Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56
–> pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls
Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56
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incubation with anti-GFP nanobody (5µl/spot, 330 µg/ml, 120 min)
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blocked with BSA(2%) (90 min)
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rinsed with PBS(1x)
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stored in PBS(1x) (for 9 days)
Spot No. | Sample |
1 | GOPTS + nanobody + B52 |
2 | GOPTS + nanobody + B53 |
3 | GOPTS + nanobody + B54 |
4 | GOPTS + nanobody + B55 |
5 | GOPTS + nanobody + B56 |
6 | GOPTS + GFP (positive control) |
7 | GOPTS + WT (negative control) |
07/15/2016
Sample preparation
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added nanobody to GOPTS plate (300 µg/ml, 5µl)
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tried to pipette rest nanobody down, but didn´t work
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blocked complete slide with BSA(2%)
07/23/016
Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56
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washed slides with ddH2O
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incubation with B52, B53, B54, B55, B56 (50µl/spot, overnight)
–> FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP
07/24/2016
Preparation of samples 3: B53-B55
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B53, B54, B55 (10µl/spot) on uncoated glass slide
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Mowiol DABCO (5µl/spot)
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covered with cover slip
Spot No. | Sample |
1 | B52 |
2 | B53 |
3 | B54 |
Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56
–> fluorescence signal of B53 and B54
Fluorescence microscopy 3 (Olympus 1×71): B53-B55
–> fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
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incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d)
07/26/2016
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
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blocked with BSA(4%) (overnight)
07/27/2016
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
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rinsed and washed with PBS(1x) (10 min, 10rpm)
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rinsed with water ddH2O
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dried with compressed air
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incubation with Mowiol DABCO (5µl/spot, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (positive control) |
2 | GOPTS + nanobody + GFP |
3 | GOPTS + BSA + GFP (negative control) |
4 | GOPTS + B53 (positive control) |
5 | GOPTS + B54 (positive control) |
6 | B53 (positive control) |
7 | B54 (positive control) |
07/28/2016
Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54
–> spores appear to not have been bound to nanobody
08/08/2016
Sample preparation 5: GOPTS + anti-GFP nanobody + B53-56/GFP
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incubation with anti-GFP nanobody (5µl, 330µg/ml, 60 min)
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rinsed and blocked with BSA(4%) (30 min)
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incubation with B53, B54, B55, B56, GFP (5µl/spot, 60 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with water ddH2O
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dried with compressed air
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incubation with Mowiol DABCO (5µl/spot, overnight)
Spot No. | Sample |
1 | GOPTS + nanobody + B55 (negative control) |
2 | GOPTS + B55 (negative control) |
3 | GOPTS + nanobody + B53 |
4 | GOPTS + nanobody + GFP (positive control) |
5 | GOPTS + BSA + GFP (negative control) |
6 | B53 (positive control) |
7 | B54 (positive control) |
8 | GOPTS + GFP (positive control) |
9 | GOPTS + B53 (positive control) |
10 | GOPTS + nanobody(Alexa647) + B53 |
11 | GOPTS + nanobody(Alexa647) + B54 |
12 | GOPTS + nanobody(Alexa647) + WT(negative control) |
13 | GOPTS + nanobody(Alexa647) + B56 (negative control) |
08/09/2016
Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP
–> spores have not been bound
08/10/2016
Sample preparation 6: anti-GFP nanobody + spores (WT, B53, B54, B56) (in solution)
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incubation of anti-GFP nanobody (Alexa647-conjugated) (1µl, 330µg/ml, 60 min) + spores (WT, B53, B54, B56) (400µl, 60 min, 4°C)
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centrifuged (13400 rpm, 2 min)
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washed with PBS(1x) (1 ml)
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repeated steps 2 and 3
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centrifuged (13400 rpm, 2 min)
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resuspended in PBS(1x) (400µl)
Spot No. | Sample |
1 | nanobody + WT (negative control) |
2 | nanobody + B53 |
3 | nanobody + B53 |
4 | nanobody + B55 (negative control) |
5 | nanobody + B56(negative control) |
6 | nanobody + GFP (positive control) |
7 | nanobody (positive control) |
8 | GFP (positive control) |
Fluorescence microscopy 6 (Nikon C2+ confocal microscope): anti-GFP nanobody + spores (WT, B53, B54, B56) in solution
–> no introduction to confocal mode: nanobody not observable
08/14/2016
Sample preparation 7: GOPTS + anti-GFP nanobody + B53/B54
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incubated with nanobody (1µl, 330µg/ml, 60 min)
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rinsed and blocked with BSA(4%) (60 min)
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rinsed and washed twice with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with spores (10µl/spot, 120 min)
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rinsed and washed twice with PBS(1x) (10 min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | nanobody + WT (negative control) |
2 | nanobody + B53 |
3 | nanobody + B54 |
4 | nanobody + GFP (positive control) |
5 | nanobody (positive control) |
6 | GFP (positive control) |
Washing test A: GOPTS + anti-GFP nanobody + B53
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incubation with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)
08/15/2016
Washing test A: GOPTS + anti-GFP nanobody + B53
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rinsed and blocked with BSA(2%) (60 min, 10 rpm)
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rinsed with PBS(1x), washed PBS twice (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubation with B53 (10µl/spot, overnight)
08/16/2016
Washing test A: GOPTS + anti-GFP nanobody + B53
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rinsed and washed (see table below)
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rinsed with ddh2O
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample | Condition |
1 | GOPTS + nanobody + B53 | washed 2x with PBS(1x) (10 min, 10 rpm) |
2 | GOPTS + nanobody + B53 | washed 3x with PBS(1x) (10 min, 10 rpm) |
3 | GOPTS + nanobody + B53 | washed 4x with PBS(1x) (10 min, 10 rpm) |
4 | GOPTS + nanobody + B53 | washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm) |
5 | GOPTS + nanobody + B53 | washed 1x with PBS(1x) + 0,1 % Tween20 (10 min, 10 rpm) |
08/17/2016
Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54
–> spores have not bound to nanobody
Fluorescence microscopy A (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53
–> spores have not bound to BSA
08/21/2016
Sample preparation 8: GOPTS + anti-GFP nanobody + GFP
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incubation with nanobody (see table below, 60 min)
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rinsed and blocked with BSA(4%) (60 min)
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rinsed and washed with PBS(1x) (5 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with GFP (see table below, 1.35mg/ml, 20 min)
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rinsed and washed with PBS(1x) (10 min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (1µl) (positive control) |
2 | GOPTS + GFP (5 µl) (positive control) |
3 | GOPTS + nanobody (1µl, 330µg/ml) + GFP |
4 | GOPTS + nanobody (0.5µl, 10mg/ml) + GFP |
5, 6 | GOPTS + BSA + GFP (negative control) |
08/22/2016
Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
–> GFP not bound, GOPTS plates too old
08/23/2016
Sample preparation 9: GOPTS + GFP
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incubation with GFP (1.35mg/ml, volume/incubation: see table below)
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rinsed and blocked with BSA(4%) (30 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (1 µl, 10 min) |
2 | GOPTS + GFP (2 µl, 10 min) |
3 | GOPTS + GFP (5 µl, 10 min) |
4 | GOPTS + GFP (1 µl, 15 min) |
5 | GOPTS + GFP (2 µl, 15 min) |
6 | GOPTS + GFP (5 µl, 15 min) |
7 | GOPTS + GFP (1 µl, 20 min) |
8 | GOPTS + GFP (2 µl, 20 min) |
9 | GOPTS + GFP (5 µl, 20 min) |
10 | GOPTS (negative control) |
Sample preparation 10: GOPTS + anti-GFP nanobody + B53
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incubation with nanobody (1µl, 330µg/ml, 60 min)
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rinsed and blocked with BSA(4%) (60 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with spores (10µl/spot, 30 min)
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rinsed and washed with PBS(1x) (10min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + nanobody + B53 |
2 | GOPTS + nanobody + B53 |
3 | GOPTS + GFP(positive control) |
4 | GOPTS + nanobody + GFP (positive control) |
5 | GOPTS + BSA + B53 (negative control) |
Washing test B: GOPTS + anti-GFP nanobody + B53
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incubation with nanobody (1µl, 330µg/ml, 60 min)
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rinsed and blocked with BSA(4%) (60 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with spores (10µl/spot, 30 min)
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rinsed and washed with PBS(1x) (10 min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample | Condition |
1 | GOPTS + nanobody + B53 | rinsed with PBS(1x) |
2 | GOPTS + nanobody + B53 | swivel with PBS(1x) (0.5 min) |
3 | GOPTS + nanobody + B53 | washed with PBS(1x) (1 min, 10 rpm) |
4 | GOPTS + nanobody + B53 | washed with PBS(1x) (2 min, 10 rpm) |
5 | GOPTS + nanobody + B53 | washed with PBS(1x) (3 min, 10 rpm) |
6 | GOPTS + nanobody + B53 | washed with PBS(1x) (5 min, 10 rpm) |
7 | GOPTS + BSA + B53(negative control) | washed with PBS(1x) (10 min, 10 rpm) |
8 | GOPTS + BSA + GFP(negative control) | washed with PBS(1x) (10 min, 10 rpm) |
9 | GOPTS + GFP (positive control) | washed with PBS(1x) (10 min, 10 rpm) |
10 | GOPTS + B53 (positive control) | washed with PBS(1x) (10 min, 10 rpm) |
08/24/2016
Fluorescence microscopy 9 (Nikon C2+ confocal microscope): GOPTS + GFP
–> strong fluorescence signal at some of the edges: incubation too long/volume too little, GFP has dried and could not have been washed away properly
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1) GOPTS + GFP (1 µl, 10 min)
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2) GOPTS + GFP (2 µl, 10 min)
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3) GOPTS + GFP (5 µl, 10 min)
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4) GOPTS + GFP (1 µl, 15 min)
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5) GOPTS + GFP (2 µl, 15 min)
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6) GOPTS + GFP (5 µl, 15 min)
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7) GOPTS + GFP (1 µl, 20 min)
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8) GOPTS + GFP (2 µl, 20 min)
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9) GOPTS + GFP (5 µl, 20 min)
Fluorescence microscopy 10 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53
–> no spores appear to have bound to nanobody
Fluorescence microscopy B (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53
–> no spores after washing, no binding between nanobody and B53?
08/25/2016
Blocking test C: GOPTS + BSA/milk powder + GFP
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blocking with BSA(4%)/milk powder(5%) (120 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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incubation with GFP (1µl/spot, 1.35mg/ml, 20 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH20
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dried with compressed air
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incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (positive control) |
2 | GOPTS + milk powder(5%) + GFP |
3 | GOPTS + BSA(4%) + GFP |
Contamination Test D: BSA, PBS Mowiol DABCO
–> no contamination/pollution detected
Sample preparation 11: GOPTS + mCherry
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incubated with mCherry (1µl, 2mg/ml, incubation: see table below)
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rinsed and blocked with BSA(4%) (30 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + mCherry (5 min) |
2 | GOPTS + mCherry (10 min) |
3 | GOPTS + mCherry (15 min) |
4 | GOPTS (negative control) |
5 | mCherry + Mowiol DABCO (positive control) |
Sample preparation 12: GOPTS + anti-GFP nanobody + GFP
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incubated with nanobody (5µl, 330µg/ml, 120 min)
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rinsed and blocked with milk powder(5%) (60 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with GFP (2µl, 1.35mg/ml, 20 min)
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rinsed and washed with PBS(1x) (10 min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + nanobody + GFP |
2 | GOPTS (negative control) |
3 | GOPTS + GFP (positive control) |
4 | GOPTS + nanobody (negative control) |
5 | GOPTS + BSA + GFP (negative control) |
08/27/2016
Fluorescence microscopy C (Nikon C2+ confocal microscope): Blocking test: GOPTS + BSA/milkpowder + GFP
–> No GFP signal after blocking with BSA and milk powder
Fluorescence microscopy 11 (Nikon C2+ confocal microscope): GOPTS + mCherry
–> no mCherry-signal, too weak (quantum yield of mCherry about 1/3 of that of GFP)
Fluorescence microscopy 12 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
–> GFP has not bound to nanobody
08/30/2016
Sample preparation 13: GOPTS + nanobody + GFP
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incubated with nanobody (5µl, 330µg/ml, 60 min)
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rinsed and blocked with milk powder(5%) (30 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
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incubated with GFP (2µl, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1,2 | GOPTS + nanobody(Alexa647) + GFP |
3,4 | GOPTS + nanobody + GFP |
5 | GOPTS + GFP (positive control) |
6 | GOPTS + nanobody (negative control) |
7 | GOPTS + BSA (negative control) |
09/01/2016
Fluorescence microscopy 13 (Nikon C2+ confocal microscope): GOPTS + nanobody + GFP
–> no GFP signal detectable
09/06/2016
Sample preparation 14: GOPTS + GFP
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incubated with GFP (10µl, 1.35mg/ml, 30 min)
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rinsed and blocked with milk powder(5%) (30 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1,2 | GOPTS + mCherry |
3 | GOPTS + BSA + mCherry (negative control) |
4 | GOPTS + BSA (negative control) |
5 | mCherry (positive control) |
Mowiol was produced due to this protocol: https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf
09/07/2016
Fluorescence microscopy 14 (Nikon C2+ confocal microscope): GOPTS + GFP
–> spots with evenly distributed GFP
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1) GOPTS + GFP
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2) GOPTS + nanobody + GFP
09/14/2016
Sample preparation 16: GOPTS + B53
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incubated with B53 (5µl, 60 min)
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rinsed and blocked with milk powder(4%) (30 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1,2,3 | GOPTS + B53 |
4 | GOPTS + WT (negative control) |
5 | WT (negative control) |
6 | B53 (positive control) |
7 | GOPTS + GFP |
09/15/2016
Sample preparation 17: GOPTS + anti-GFP nanobody(Alexa647) + GFP
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incubated with nanobody (5µl, 330µg/ml, 60 min)
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rinsed and blocked with milk powder(5%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with GFP (2µl, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1-4 | GOPTS + nanobody(Alexa647) + GFP |
5 | GOPTS + milk powder + GFP (negative control) |
6 | GOPTS + milk powder + NB (negative control) |
7 | GOPTS + GFP |
8 | GOPTS (negative control) |
Fluorescence microscopy 16 (Nikon C2+ confocal microscope): GOPTS + B53
–> no spores detectable
09/16/2016
Fluorescence microscopy 17 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
–> GFP signal: GFP has been bound to nanobody
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1-4) GOPTS + nanobody(Alexa647) + GFP
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5) GOPTS + milk powder + GFP (negative control)
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6) GOPTS + milk powder + NB (negative control)
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7) GOPTS + GFP (positive control)
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8) GOPTS (negative control)
09/17/2016
Sample preparation 18: GOPTS + anti-GFP nanobody + GFP (spotting-mask)
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incubation with anti-GFP nanobody ( 2µl/spot, 330µg/ml, 10 min, 4°C)
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rinsing and blocking with milk powder(5%) (15 min, 10 rpm)
-
rinsing with PBS(1x)
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incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
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(removed mask)
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rinsed with ddH2O
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dried with compressed air
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incubated with Mowiol DABCO (overnight)
09/18/2016
Fluorescence microscopy 18 (Nikon C2+ confocal microscope): GOPTS + antiGFP-nanobody + GFP (spotting-mask)
–> no GFP signal detectable
Sample preparation 19: GOPTS + anti-GFP nanobody + GFP (spotting-mask)
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incubation with anti-GFP nanobody (2µl/spot, 330µg/ml, 15 min, 4°C)
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rinsing with PBS(1x)
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(removed mask)
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rinsing and blocking with milk powder(5%) (15 min, 2 rpm)
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(reattached mask)
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incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
-
(removed mask)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (overnight)
09/19/2016
Fluorescence microscopy 19 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP (spotting-mask)
–> slight GFP-signal on nanobody
Sample preparation 20: anti-GFP nanobody + B53 (in solution)
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incubation of anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 15 min, 4°C) + B53
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incubated on GOPTS-slide (2µl, 15 min)
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rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
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rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)
–> problem: no washing away of unbound nanobody
Spot No. | Sample |
1,2 | B53 + nanobody(Alexa647) (incubated in solution) on GOPTS |
3,4 | WT + nanobody(Alexa647) (incubated in solution) on GOPTS (negative control) |
5,6 | B53 + nanobody(Alexa647) (incubated in solution) dried on milk powder |
7,8 | WT + nanobody(Alexa647) (incubated in solution) dried on milk powder (negative control) |
9,10 | GOPTS + nanobody(Alexa647) + GFP (positive control) |
11,12 | GOPTS + nanobody(Alexa647) + B53 |
13,14 | GOPTS + nanobody(Alexa647) + WT (negative control) |
15 | GOPTS + milk powder + nanobody(Alexa647) (negative control) |
16 | GOPTS + milk powder + B53 (negative control) |
17 | GOPTS + milk powder + WT (negative control) |
18 | GOPTS + GFP (positive control) |
19 | GOPTS + nanobody(Alexa647) (negative control) |
20 | B53 dried (positive control) |
09/20/2016
Sample preparation 21: anti-GFP nanobody + B53 (in solution)
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incubation with anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 30 min, 4°C) + B53
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centrifuged (2 min, 13400 rpm)
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washed with PBS(1x)
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repeated steps 2 and 3
-
resuspended in PBS(1x) (30µl)
-
incubated on GOPTS-slide (2µl, 15 min)
-
rinsed and blocked with milkpowder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)
Spot No. | Sample |
1 | B53 + nanobody(Alexa647) (incubated in solution) on GOPTS |
2 | WT + nanobody(Alexa647) (incubated in solution) on GOPTS |
3,4 | B53 + nanobody(Alexa647) dried on milkpowder |
5,6 | WT + nanobody(Alexa647) dried on milkpowder |
09/29/2016
Sample preparation 22: GOPTS + GFP
-
incubation with GFP (1 µl, 1.35mg/ml, 30 min)
-
rinsed and blocked with milk powder(5%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1-5 | GOPTS + GFP |
6 | GOPTS |
09/30/2016
–> Construct spores are ready for analysis
Strain No. | Strain |
3 | 150: aGFPnano_HA_aHelix_cgeA |
6 | 151: aGFPnano_HA_aHelix_cotG |
7 | 159: aGFPnano_HA_G4S_CotZ |
9 | 157: aGFPnano_HA_aHelix_CotZ |
Fluorescence microscopy 22 (Nikon C2+ confocal microscope): GOPTS + GFP
–> spots with evenly distributed GFP
-
incubation with GFP (1 µl, 1.35mg/ml, 30 min)
-
spores centrifuged (2 min, 13,400 rpm)
-
washed with PBS(1x)
-
step 1 repeated
-
resuspended in PBS(1x)
-
incubation on GOPTS-slide(GFP) with spores (4µl, 400,000/µl, incubation time: see table below, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Incubation time (min) |
1 | GOPTS + GFP + strain 3 | 5 |
2 | GOPTS + GFP + strain 3 | 10 |
3 | GOPTS + GFP + strain 3 | 15 |
4 | GOPTS + GFP + strain 6 | 5 |
5 | GOPTS + GFP + strain 6 | 10 |
6 | GOPTS + GFP + strain 6 | 15 |
7 | GOPTS + GFP + strain 7 | 5 |
8 | GOPTS + GFP + strain 7 | 10 |
9 | GOPTS + GFP + strain 7 | 15 |
10 | GOPTS + GFP + strain 9 | 5 |
11 | GOPTS + GFP + strain 9 | 10 |
12 | GOPTS + GFP + strain 9 | 15 |
13, 14 | GOPTS + GFP (positive control) | 10 |
15 | GOPTS + GFP + WT (negative control) | 5 |
16 | GOPTS + GFP + WT (negative control) | 10 |
17 | GOPTS + GFP + WT (negative control) | 15 |
18 | GOPTS + strain 3 (positive control) | 10 |
19 | GOPTS + strain 6 (positive control) | 10 |
20 | GOPTS + strain 7 (positive control) | 10 |
21 | GOPTS + strain 9 (positive control) | 10 |
22 | GOPTS + WT (negative control) | 10 |
10/01/2016
Sample preparation 23: GOPTS + GFP + spores
–> no spores detectable
Sample preparation 24: GOPTS + GFP + spores (in EtOH resuspended)
-
incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (10 min, 4°C)
-
solution centrifuged (4 min, 13,400 rpm)
-
washed with PBS(1x)
-
step 2 repeated
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample |
1-3 | GOPTS + GFP + strain 6 |
4-6 | GOPTS + GFP + WT |
7-9 | GOPTS + GFP |
10-12 | GOPTS + strain 6 |
13-15 | GOPTS + WT |
10/02/2016
Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (strains 3,6,7,9,wt) (in EtOH resuspended)
–> EtOH denaturated GFP: very weak signal. No spores detectable
Sample preparation 25: GFP + spores (strains 3,6,7,9) (in solution)
-
incubation of GFP (4µl, 1,35g/ml) + spores(strain 3,6,7,9) (4µl, 400,000/µl) (incubation time: see table below, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3
-
resuspended in PBS(1x) (20µl)
-
incubated on GOPTS-slide (4µl, 10 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubrnight)ation with Mowiol DABCO
Spot No. | Sample |
1, 2 | GFP + strain 3 |
3, 4 | GFP + strain 6 |
5, 6 | GFP + strain 7 |
7, 8 | GFP + strain 9 |
9, 10 | GFP + WT (negative control) |
11, 12 | GFP + PBS (positive control) |
13, 14 | GFP (positive control) |
15, 16 | WT (negative control) |
17 | strain 3 (negative control) |
18 | strain 6 (negative control) |
19 | strain 7 (negative control) |
20 | strain 9 (negative control) |
10/03/2016
Sample preparation 26: Incubation test: GFP + spores (in solution)
-
incubation of GFP (4µl, 1,35g/ml) + spores (4µl, 400,000/µl) (incubation time: see tables below, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3)
-
resuspended in PBS(1x) (20µl))
-
incubated on GOPTS-slide (4µl, 10 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Slide 1:
Spot No. | Sample | Incubation time |
1, 2 | GFP + strain 3 | 30 min |
3, 4 | GFP + strain 3(pellet washed with PBS(1x)) | 30 min |
5, 6 | GFP + strain 6 | 30 min |
7, 8 | GFP + strain 6(pellet washed with PBS(1x)) | 30 min |
9, 10 | GFP + strain 7 | 30 min |
11, 12 | GFP + strain 7(pellet washed with PBS(1x)) | 30 min |
13, 14 | GFP + strain 9 | 30 min |
15, 16 | GFP + strain 9 (pellet washed withn PBS(1x)) | 30 min |
17, 18 | GFP + WT (negative control) | |
19, 20 | GFP + WT (negative control) (pellet washed with PBS(1x)) | |
21, 22 | GFP (positive control) | |
23, 24 | WT (negative control) | |
25 | strain 3 (negative control) | |
26 | strain 6 (negative control) | |
27 | strain 7 (negative control) | |
28 | strain 9 (negative control) |
Slide 2:
Spot No. | Sample | Incubation time |
1, 2 | GFP + strain 3 | 60 min |
3, 4 | GFP + strain 3(pellet washed with PBS(1x)) | 60 min |
5, 6 | GFP + strain 6 | 60 min |
7, 8 | GFP + strain 6(pellet washed with PBS(1x)) | 60 min |
9, 10 | GFP + strain 7 | 60 min |
11, 12 | GFP + strain 7(pellet washed with PBS(1x)) | 60 min |
13, 14 | GFP + strain 9 | 60 min |
15, 16 | GFP + strain 9 (pellet washed withn PBS(1x)) | 60 min |
17, 18 | GFP + WT (negative control) | |
19, 20 | GFP + WT (negative control) (pellet washed with PBS(1x)) | |
21, 22 | GFP (positive control) | |
23, 24 | WT (negative control) | |
25 | strain 3 (negative control) | |
26 | strain 6 (negative control) | |
27 | strain 7 (negative control) | |
28 | strain 9 (negative control) |
Slide 3:
Spot No. | Sample | Incubation time |
1, 2 | GFP + strain 3 | 120 min |
3, 4 | GFP + strain 3(pellet washed with PBS(1x)) | 120 min |
5, 6 | GFP + strain 6 | 120 min |
7, 8 | GFP + strain 6(pellet washed with PBS(1x)) | 120 min |
9, 10 | GFP + strain 7 | 120 min |
11, 12 | GFP + strain 7(pellet washed with PBS(1x)) | 120 min |
13, 14 | GFP + strain 9 | 120 min |
15, 16 | GFP + strain 9 (pellet washed withn PBS(1x)) | 120 min |
17, 18 | GFP + WT (negative control) | |
19, 20 | GFP + WT (negative control) (pellet washed with PBS(1x)) | |
21, 22 | GFP (positive control) | |
23, 24 | WT (negative control) | |
25 | strain 3 (negative control) | |
26 | strain 6 (negative control) | |
27 | strain 7 (negative control) | |
28 | strain 9 (negative control) |
Sample preparation 28: GFP-volume test B: GFP + strain 7 (in solution, washed)
-
incubation of GFP (volume: see table below, 1,35g/ml) + strain 7 (4µl, 400,000/µl) (90 min, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3)
-
resuspended in PBS(1x) (20µl)
-
incubated on GOPTS-slide (2µl, 90 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Volume (GFP) |
1, 2 | GFP + strain 7 | 0,5µl |
3, 4 | GFP + strain 7 | 1,0µl |
5, 6 | GFP + strain 7 | 1,5µl |
7, 8 | GFP + strain 7 | 2,0µl |
9, 10 | GFP + strain 7 | 2,5µl |
11, 12 | GFP (positive control) | 4,0µl |
13, 14 | strain 7 (negative control) | |
15, 16 | GFP + WT (negative control) | 0,5µl |
17, 18 | GFP + WT (negative control) | 1,0µl |
19, 20 | GFP + WT (negative control) | 1,5µl |
21, 22 | GFP + WT (negative control) | 2,0µl |
23, 24 | GFP + WT (negative control) | 2,5µl |
25, 26 | WT (negative control) |
Sample preparation 29: Spore-amount test: GFP + strain 7 (in solution)
-
incubation of GFP (0,5µl, 1,35g/ml) + strain 7 (amount: see table below, 400,000/µl) (90 min, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3)
-
resuspended in PBS(1x) (20µl)
-
incubated on GOPTS-slide (2µl, 90 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Amount (spores) |
1, 2 | strain 7 (negative control) | 25,000,000 |
3, 4 | strain 7 (negative control) | 15,000,000 |
5, 6 | strain 7 (negative control) | 10,000,000 |
7, 8 | GFP + strain 7 | 25,000,000 |
9, 10 | GFP + strain 7 | 15,000,000 |
11, 12 | GFP + strain 7 | 10,000,000 |
13, 14 | GFP (positive control) | |
15, 16 | WT (negative control) | 25,000,000 |
17, 18 | WT (negative control) | 15,000,000 |
19, 20 | WT(negative control) | 10,000,000 |
21, 22 | GFP + WT (negative control) | 25,000,000 |
23, 24 | GFP + WT (negative control) | 15,000,000 |
25, 26 | GFP + WT (negative control) | 10,000,000 |
10/04/2016
Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (in EtOH resuspended)
–> spore concentration too high, too many layers of spores
Fluorescence microscopy 25 (Nikon C2+ confocal microscope): GFP + spores (in solution)
–> spore concentration too high, too many layers of spores
Fluorescence microscopy 26 (Nikon C2+ confocal microscope): Incubation test: GFP + spores (in solution)
–> spore concentration too high, too many layers of spores
Slide 1:
1.1) strain7 + GFP (30 min) (white field) 1.2) strain7 + GFP (30 min) (GFP-channel) 2.1) strain9 + GFP (30 min) (white field) 2.2) strain9 + GFP (30 min) (GFP-channel)
Slide 2:
1.1) strain 7 + GFP (90 min)(white field) 1.2) strain 7 + GFP (90 min)(GFP-channel) 2.1) strain WT + GFP (90 min)(white field) 2.2) strain WT + GFP (90 min)(GFP-channel)
Fluorescence microscopy 27 (Nikon C2+ confocal microscope): GFP-volume test A: GFP + strain 7 (in solution)
–> background fluorescence was too high
Fluorescence microscopy 28 (Nikon C2+ confocal microscope): GFP-volume test B: GFP + strain 7 (in solution)
–> background fluorescence was too high
Fluorescence microscopy 29 (Nikon C2+ confocal microscope): GFP + strain 7 (in solution)
–> spores and WT show similar levels of fluorescence, probably autofluorescence
1) strain 7 + GFP (white field) 2) strain 7 + GFP (GFP-channel) 3) WT + GFP (white field) 4) WT + GFP (GFP-channel)
Sample preparation 30: Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)
-
incubation of GFP (1µl, 1,35g/ml) + spores (strain 3,6,7,9) (25,000.000 spores) (90 min, 4°C)
-
solution centrifuged (2 min, 13,400 rpm)
-
washed with PBS(1x)
-
step 2 and 3 repeated (2x/4x)
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Washing of GFP-spore solution |
1, 2 | GFP + strain 3 | 2x |
3, 4 | GFP + strain 6 | 2x |
5, 6 | GFP + strain 7 | 2x |
7, 8 | GFP + strain 9 | 2x |
9, 10 | GFP + WT (negative control) | 2x |
11, 12 | GFP + strain 3 | 4x |
13, 14 | GFP + strain 6 | 4x |
15, 16 | GFP + strain 7 | 4x |
17, 18 | GFP + strain 9 | 4x |
19, 20 | GFP + WT (negative control) | 4x |
21, 22 | GFP (positive control) | |
23 | WT (negative control) | |
24 | strain 3 (negative control) | |
25 | strain 6 (negative control) | |
26 | strain 7 (negative control) | |
27 | strain 9 (negative control) |
Sample preparation 31: Wash-test B: GFP + spores (in solution)
-
incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
-
solution centrifuged (2 min, 13,400 rpm)
-
washed with TBST(0,005%)
-
steps 2 and 3 repeated (2x/4x)
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Washing of GFP-spore solution |
1, 2 | GFP + strain 3 | 2x |
3, 4 | GFP + strain 6 | 2x |
5, 6 | GFP + strain 7 | 2x |
7, 8 | GFP + strain 9 | 2x |
9, 10 | GFP + WT (negative control) | 2x |
11, 12 | GFP + strain 3 | 4x |
13, 14 | GFP + strain 6 | 4x |
15, 16 | GFP + strain 7 | 4x |
17, 18 | GFP + strain 9 | 4x |
19, 20 | GFP + WT (negative control) | 4x |
21, 22 | GFP (positive control) | |
23 | WT (negative control) | |
24 | strain 3 (negative control) | |
25 | strain 6 (negative control) | |
26 | strain 7 (negative control) | |
27 | strain 9 (negative control) |
10/05/2016
Fluorescence microscopy 30 (Nikon C2+ confocal microscope): Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)
–> spore concentration too high, background fluorescence too high
Fluorescence microscopy 31 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (strains 3,6,7,9) (in solution)
–> indicates that strain 3 binds GFP
-
1.1) GFP + strain 3 (white field)
-
1.2) GFP + strain 3 (GFP-channel)
-
2.1) GFP + WT (negative control) (white field)
-
2.2) GFP + WT (negative control) (GFP-channel)
-
3) WT (negative control)
10/06/2016
Sample preparation 32: Wash-test C: GFP + spores (in solution)
-
incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
-
solution centrifuged (2 min, 13,400 rpm)
-
washed with TBST(0,005%)
-
steps 2 and 3 repeated (1x/3x/5x)
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Washing of GFP-spore solution (step 3) |
1, 2 | GFP + strain 3 | 1x |
3, 4 | GFP + strain 7 | 1x |
5, 6 | GFP + WT | 1x |
7, 8 | GFP + strain 3 | 3x |
9, 10 | GFP + strain 7 | 3x |
11, 12 | GFP + WT | 3x |
13, 14 | GFP + strain 3 | 5x |
15, 16 | GFP + strain 7 | 5x |
17, 18 | GFP + WT | 5x |
19, 20 | WT (negative control) | |
21, 22 | GFP (positive control) |
10/07/2016
Fluorescence microscopy 32 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (in solution)
–> spores and WT show similar levels of fluorescence, probably autofluorescence
Sample preparation 33: Blocking-test A: spores milk powder + GFP (in solution)
-
solution centrifuged (2 min, 13,400 rpm)
-
spores washed twice with PBS(1x) (2x 100µl)
-
spores resuspended and incubated in milk powder(5%) (100µl, incubation time: see table below)
-
incubation with GFP (5µl, 1,35g/ml, 90 min)
-
washed with PBS(1x) (3x 100µl)
-
resuspended in PBS(1x) (20 µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Incubation time |
1, 2 | strain 3 + milk powder + GFP | 30 min |
3, 4 | strain 3 + milk powder + GFP | 60 min |
5, 6 | strain 7 + milk powder + GFP | 30 min |
7, 8 | strain 7 + milk powder + GFP | 60 min |
9, 10 | WT + milk powder + GFP (negative control) | 30 min |
11, 12 | WT + milk powder + GFP(negative control) | 60 min |
13, 14 | WT + milk powder (negative control) | 30 min |
15, 16 | WT + milk powder (negative control) | 60 min |
17, 18 | strain 3 + milk powder (negative control) | 30 min |
19, 20 | strain 3 + milk powder (negative control) | 60 min |
21, 22 | strain 7 + milk powder (negative control) | 30 min |
23, 24 | strain 7 + milk powder (negative control) | 60 min |
25, 26 | GFP (positive control) | 30 min |
10/08/2016
Fluorescence microscopy 33 (Nikon C2+ confocal microscope): Blocking-test A: spores + milk powder + GFP (in solution)
–> spores and WT show similar levels of fluorescence
10/11/2016
Sample preparation 34: Blocking-test B: spores + BSA + GFP (in solution)
-
solution centrifuged (2 min, 13,400 rpm)
-
spores (5,000,000) washed with PBS(1x) (3x 100µl)
-
spores resuspended and incubated in BSA(5%) (100µl, 60 min)
-
incubation with GFP (50µl, 100µg/ml, 60 min)
-
washed with TBST(0.05%) (5x 100µl)
-
resuspended in PBS(1x) (50 µl)
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample |
1, 2 | strain 3 + BSA + GFP |
3, 4 | strain 7 + BSA + GFP |
5, 6 | WT + BSA + GFP (negative control) |
7, 8 | WT + BSA (negative control) |
9, 10 | strain 3 + milk powder (negative control) |
11, 12 | strain 7 + milk powder (negative control) |
13, 14 | GFP (positive control) |
10/12/2016
Fluorescence microscopy 34 (Nikon C2+ confocal microscope): Blocking-test B: spores + BSA + GFP (in solution)
–> too little spores