Members:Kevin, Christian, Vivi, Nathalie

Preparation of overnight cultures of 5 different Bacillus subtilis strains with GFP on CotZ-fusionprotein

Medium: Preparation of LB-medium with Chloramphenicol: 50µl(1000X) in 50ml LB-Medium

Inoculation: 5 reaction tubes with 5 ml LB-Chloramphenicol-medium have been used. Each has been inoculated with a little piece of filter paper with spores. Cultures have been incubated at 200rpm and 37°C, 16:10Pm to 8am. No growth has been observed !

Further spore production was performed by group 2.

Preparation of GOPTS coated glass slides

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.

1. labeled with a diamond pen
2. washed with EtOH then ddH2O
3. dried slides with compressed air
4. activation of surface with plasma generator
5. incubation with GOPTS (overnight)

Buffer for anti-GFP-nanobody (10 ml)

• 61mg Tris
• 87mg NaCl
• 1ml Glycerin
• 2,5 ml Imidazole(1M)
• fill up with ddH2O to 10 ml

Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56

1. Glass slides were spotted with 0,5 µl nanobody (300µg/ml) (Spot location marked with permanent marker.)
2. Slides were incubated over night in the fridge at 4°C in darkness
3. 150µl BSA(2x) on each spot, 30 min incubation
4. washed with PBS(1x)
5. washed with 20mM imidazole in PBS(1x)
6. washed with PBS(1x)
7. washed with ddH2o
8. dried with compressed air
9. 100µl of spore solution on each slide

Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56

1. blocked with BSA(2%) (150µl/spot, 30 min)
2. washed with PBS(1x)
3. washed with imidazole (20mM) in PBS(1x)
4. washed with PBS(1x)
5. washed with ddH2O
6. dried with compressed air
7. incubation with B52-B56 (100µl/spot, 120 min)
8. incubated with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top

Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56

1. edges sealed with nail polish

Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56

–> pictures inconclusive:pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls

Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56

1. incubation with anti-GFP nanobody (5µl/spot, 330 µg/ml, 120 min)
2. blocked with BSA(2%) (90 min)
3. rinsed with PBS(1x)
4. stored in PBS(1x) (for 9 days)

Sample preparation

1. added nanobody to GOPTS plate (300 µg/ml, 5µl)
2. tried to pipette rest nanobody down, but didn´t work
3. blocked complete slide with BSA(2%)

Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56

1. Washed slides with ddH2O
2. Add spores (50µl/spot)(B52,B53,B54,B55,B65)

–> FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP

Coating of new slides with GOPTS (experiment 3)

1. according to protocol

Preparation of samples 3: B53-B55

1. B53, B54, B55 (10µl/spot) on uncoated glass slide
2. Mowiol DABCO (5µl/spot)
3. covered with cover slip

Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56

1. fluorescence signal of B53 and B54

Fluorescence microscopy 3 (Olympus 1×71): B53-B55

1. fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS

B53 sample: no fluorescence observable (Air bubble)
B54 sample: no fluorescence observable

Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54

1. incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d)

Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54

1. Prepared new BSA solution with higher concentration (4% w/v)
2. Blocked slides with BSA (4%) ,covered complete slide with BSA
3. inkubated over night

Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54

1. rinsed and washed with PBS(1x) (10 min, 10rpm)
2. rinsed with water ddH2O
3. dried with compressed air
4. incubated with Mowiol DABCO (5µl/spot, overnight)

*Positive controls:

1. Slide with spores B53–> fuorescence observable
2. B54 –>Fluorescence observable

Slides of experiment 3

1. Washing with PBS
2. rinsed with water ddH2O
3. dried with compressed air

Positive controls exp. 3:

• GFP direct on GOPTS
• GFP on nanobody
• GFP on BSA blocked surface
• Spores direct on GOPTS
• double concentrated spores direct on glass slide B53 and B54

Spores were added to glass surface and incubated until its dry

Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54

–>spores appear to not have bound to nanobody

1. Washed slides of exp 3 with PBS
2. added DABCO (5µl)

prepared samples for fluorescence microscopy

Sample preparation 5: GOPTS + anti-GFP nanobody + B53-56/GFP

1. incubation with anti-GFP nanobody (5µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (30 min)
3. incubation with B53, B54, B55, B56, GFP (5µl/spot, 60 min)
4. rinsed and washed with PBS(1x) (10 min, 10 rpm)
5. rinsed with water ddH2O
6. dried with compressed air
7. incubated with Mowiol DABCO (5µl/spot, overnight)

Test if spores bind Nanobody in solution

1. Wt, B53, B54, B56 centrifuged, resuspended in 50 µl nanobody-buffer,
2. add 1µl labeled nanobody
3. incubated 1h at 4°C
4. washed 2 times 10 min with PBS

Problem: no introduction in the confocal microscopy mode

• Flourescence microscopy

Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP

–>only fluorescence detectable on slides 6 and 8

Sample preparation 6: anti-GFP nanobody + spores (WT, B53, B54, B56) (in solution)

1. incubation of anti-GFP nanobody (Alexa647-conjugated) (1µl, 330µg/ml, 60 min) + spores (WT, B53, B54, B56) (400µl, 60 min, 4°C)
2. centrifuged (13400 rpm, 2 min)
3. washed with PBS(1x) (1 ml)
4. repeated steps 2 and 3
5. centrifuged (13400 rpm, 2 min)
6. resuspended in PBS(1x) (400µl)

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

Light microscopy Analyse the slides from 28.7.2016 under light mikroscope

Sample preparation 7: GOPTS + anti-GFP nanobody + B53/B54

1. incubated with nanobody (1µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed twice with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with spores (10µl/spot, 120 min)
7. rinsed and washed twice with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubated with Mowiol DABCO (5µl, overnight)

Washing test: We want to test if spores (B53) really bind to BSA surface like the previous test showed. We also want to test if the washing procedure has an impact on this result.

1. 5 slides coated with Gopts
2. added 1 µl Nanobody (333µg/ml)
3. incubated over night

Washing test A: GOPTS + anti-GFP nanobody + B53

1. incubated with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)

Washing test A: GOPTS + anti-GFP nanobody + B53

Hier Bild von BSA + B53 mit GFP-signal

1. rinsed and blocked with BSA(2%) (60 min, 10 rpm)
2. rinsed with PBS(1x), washed PBS twice (10 min, 10 rpm)
3. rinsed with ddH2O
4. dried with compressed air
5. incubated with B53 (10µl/spot, overnight)

Washing test A: GOPTS + anti-GFP nanobody + B53

conditions
Coated plates were rinsed with PBS to remove the spore soluten. Slides were put in PBS in Petri dishes and put on the shaker.

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, picture of BSA + B53 must have been a mistake

Sample preparation 8: GOPTS + anti-GFP nanobody + GFP

–> 10 slides activated with plasmagenerator coated with GOPTS.

1. incubated with nanobody (see table below, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed with PBS(1x) (5 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with GFP (see table below, 1.35mg/ml, 20 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubated with Mowiol DABCO (5µl, overnight)
    

Picture after editing of shadow and contrast: Bedingungen: 5µl,GFP,20min. incubation, mit BSA blocking, 5min. waschen mit PBS,

Blocking Test We want to test if the GOPTS is properly blocked by BSA and if milkprotein is also usefull for blocking

1 Slide with :

Contamination Test To avoid wrong interpretations of the fluorescence pictures we tested the chemikals and solutions we use, to find out if there are fluorescent contaminations.

Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP

Sample preparation 9: GOPTS + GFP

1. incubated with GFP (1.35mg/ml, volume/incubation: see table below)
2. rinsed and blocked with BSA(4%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with Mowiol DABCO (5µl, overnight)

Sample preparation 10: GOPTS + anti-GFP nanobody + - incubated with nanobody (1µl, 330µg/ml, 60 min)

1. rinsed and blocked with BSA(4%) (60 min)
2. rinsed and washed with PBS(1x) (10 min, 10 rpm)
3. rinsed with ddH2O
4. dried with compressed air
5. incubated with spores (10µl/spot, 30 min)
6. rinsed and washed with PBS(1x) (10min, 10rpm)
7. rinsed with ddH20
8. dried with compressed air
9. incubated with Mowiol DABCO (5µl, overnight)
   

Washing test B: GOPTS + anti-GFP nanobody + B53

1. incubated with nanobody (1µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with spores (10µl/spot, 30 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubated with Mowiol DABCO (5µl, overnight)