Team:Freiburg/NotebookTargeting

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Group 4

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.

  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)

07/10/2016

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

07/12/2016


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

  1. incubation with nanobody (0.5µl, 330µg/ml, overnight, 4°C)

07/13/2016


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. incubation with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top
Spot No.Sample
1GOPTS + nanobody + B52
2GOPTS + nanobody + B53
3GOPTS + nanobody + B54
4GOPTS + nanobody + B55
5GOPTS + nanobody + B56


07/14/2016


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)


Spot No.Sample
1GOPTS + nanobody + B52
2GOPTS + nanobody + B53
3GOPTS + nanobody + B54
4GOPTS + nanobody + B55
5GOPTS + nanobody + B56
6GOPTS + GFP (positive control)
7GOPTS + WT (negative control)


07/15/2016


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%)

07/23/016


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

  1. washed slides with ddH2O
  2. 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

  1. B53, B54, B55 (10µl/spot) on uncoated glass slide
  2. Mowiol DABCO (5µl/spot)
  3. covered with cover slip
Spot No.Sample
1B52
2B53
3B54


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

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

07/26/2016


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

  1. blocked with BSA(4%) (overnight)

07/27/2016


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. incubation with Mowiol DABCO (5µl/spot, overnight)
Spot No.Sample
1GOPTS + GFP (positive control)
2GOPTS + nanobody + GFP
3GOPTS + BSA + GFP (negative control)
4GOPTS + B53 (positive control)
5GOPTS + B54 (positive control)
6B53 (positive control)
7B54 (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

  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. incubation with Mowiol DABCO (5µl/spot, overnight)
Spot No.Sample
1GOPTS + nanobody + B55 (negative control)
2GOPTS + B55 (negative control)
3GOPTS + nanobody + B53
4GOPTS + nanobody + GFP (positive control)
5GOPTS + BSA + GFP (negative control)
6B53 (positive control)
7B54 (positive control)
8GOPTS + GFP (positive control)
9GOPTS + B53 (positive control)
10GOPTS + nanobody(Alexa647) + B53
11GOPTS + nanobody(Alexa647) + B54
12GOPTS + nanobody(Alexa647) + WT(negative control)
13GOPTS + nanobody(Alexa647) + B56 (negative control)


08/09/2016


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

–> only fluorescence detectable on spots 6 and 8

1.)alexaNB+B54, 2.)GOPTS+B54, 3)GOPTS+GFP, 4)alexaNB+B53

08/10/2016


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)
Spot No.Sample
1nanobody + WT (negative control)
2nanobody + B53
3nanobody + B53
4nanobody + B55 (negative control)
5nanobody + B56(negative control)
6nanobody + GFP (positive control)
7nanobody (positive control)
8GFP (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

  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. incubation with Mowiol DABCO (5µl, overnight)
Spot No.Sample
1nanobody + WT (negative control)
2nanobody + B53
3nanobody + B54
4nanobody + GFP (positive control)
5nanobody (positive control)
6GFP (positive control)


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

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

08/15/2016


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

  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. incubation with B53 (10µl/spot, overnight)


08/16/2016


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

  1. rinsed and washed (see table below)
  2. rinsed with ddh2O
  3. dried with compressed air
  4. incubated with Mowiol DABCO (5µl, overnight)


Spot No.SampleCondition
1GOPTS + nanobody + B53washed 2x with PBS(1x) (10 min, 10 rpm)
2GOPTS + nanobody + B53washed 3x with PBS(1x) (10 min, 10 rpm)
3GOPTS + nanobody + B53washed 4x with PBS(1x) (10 min, 10 rpm)
4GOPTS + nanobody + B53washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm)
5GOPTS + nanobody + B53washed 1x with PBS(1x) + 0,1 % Tween20 (10 min, 10 rpm)


17/08/2016


Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54

–> spores have not been 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

08/21/2016


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

  1. incubation 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. incubation with Mowiol DABCO (5µl, overnight)
Spot No.Sample
1GOPTS + GFP (1µl) (positive control)
2GOPTS + GFP (5 µl) (positive control)
3GOPTS + nanobody (1µl, 330µg/ml) + GFP
4GOPTS + nanobody (0.5µl, 10mg/ml) + GFP
5, 6GOPTS + BSA + GFP (negative control)

1)1µlGFP auf GOPTS 2)5µlGFP auf GOPTS


08/22/2016


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

–>

08/23/2016


Sample preparation 9: GOPTS + GFP

  1. incubation 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. incubation with Mowiol DABCO (5µl, overnight)
Spot No.Sample
1GOPTS + GFP (1 µl, 10 min)
2GOPTS + GFP (2 µl, 10 min)
3GOPTS + GFP (5 µl, 10 min)
4GOPTS + GFP (1 µl, 15 min)
5GOPTS + GFP (2 µl, 15 min)
6GOPTS + GFP (5 µl, 15 min)
7GOPTS + GFP (1 µl, 20 min)
8GOPTS + GFP (2 µl, 20 min)
9GOPTS + GFP (5 µl, 20 min)
10GOPTS (negative control)

(1) (2) (3) (4) (5) (6) (7) (8) (9)


Sample preparation 10: GOPTS + anti-GFP nanobody + B53

  1. incubation 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) (10min, 10rpm)
  8. rinsed with ddH20
  9. dried with compressed air
  10. incubation with Mowiol DABCO (5µl, overnight)


Spot No.Sample
1GOPTS + nanobody + B53
2GOPTS + nanobody + B53
3GOPTS + GFP(positive control)
4GOPTS + nanobody + GFP (positive control)
5GOPTS + BSA + B53 (negative control)


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

  1. incubation 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. incubation with Mowiol DABCO (5µl, overnight)


Spot No.SampleCondition
1GOPTS + nanobody + B53rinsed with PBS(1x)
2GOPTS + nanobody + B53swivel with PBS(1x) (0.5 min)
3GOPTS + nanobody + B53washed with PBS(1x) (1 min, 10 rpm)
4GOPTS + nanobody + B53washed with PBS(1x) (2 min, 10 rpm)
5GOPTS + nanobody + B53washed with PBS(1x) (3 min, 10 rpm)
6GOPTS + nanobody + B53washed with PBS(1x) (5 min, 10 rpm)
7GOPTS + BSA + B53(negative control)washed with PBS(1x) (10 min, 10 rpm)
8GOPTS + BSA + GFP(negative control)washed with PBS(1x) (10 min, 10 rpm)
9GOPTS + GFP (positive control)washed with PBS(1x) (10 min, 10 rpm)
10GOPTS + B53 (positive control)washed with PBS(1x) (10 min, 10 rpm)


24/08/2016


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

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?

25/08/2016


Blocking test C: GOPTS + BSA/milk powder + GFP

  1. blocking with BSA(4%)/milk powder(5%) (120 min)
  2. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  3. incubation with GFP (1µl/spot, 1.35mg/ml, 20 min)
  4. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  5. rinsed with ddH20
  6. dried with compressed air
  7. incubation with Mowiol DABCO (5µl, overnight)
Spot No.Sample
1GOPTS + GFP (positive control)
2GOPTS + milk powder(5%) + GFP
3GOPTS + BSA(4%) + GFP

(1) (2) (3)

Contamination Test D: BSA, PBS Mowiol DABCO

–> no contamination/pollution detected PBS+DABCO

Sample preparation 11: GOPTS + mCherry

  1. incubated with mCherry (1µl, 2mg/ml, 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)
Spot No.Sample
1GOPTS + mCherry (5 min)
2GOPTS + mCherry (10 min)
3GOPTS + mCherry (15 min)
4GOPTS (negative control)
5mCherry + Mowiol DABCO (positive control)

GOPTS(neg Control) GOPTS+mCherry 15 min mCherry eingetrocknet (pos control)


Sample preparation 12: GOPTS + anti-GFP nanobody + GFP

  1. incubated with nanobody (5µl, 330µg/ml, 120 min)
  2. rinsed and blocked with milk powder(5%) (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 GFP (2µl, 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)
Spot No.Sample
1GOPTS + nanobody + GFP
2GOPTS (negative control)
3GOPTS + GFP (positive control)
4GOPTS + nanobody (negative control)
5GOPTS + 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 clear 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


30/08/2016


Sample preparation 13: GOPTS + nanobody + GFP

  1. incubated with nanobody (5µl, 330µg/ml, 60 min)
  2. rinsed and blocked with milk powder(5%) (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 GFP (2µl, 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)
Spot No.Sample
1,2GOPTS + nanobody(Alexa647) + GFP
3,4GOPTS + nanobody + GFP
5GOPTS + GFP (positive control)
6GOPTS + nanobody (negative control)
7GOPTS + 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

  1. incubated with GFP (10µl, 1.35mg/ml, 30 min)
  2. rinsed and blocked with milk powder(5%) (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)


Spot No.Sample
1,2GOPTS + mCherry
3GOPTS + BSA + mCherry (negative control)
4GOPTS + BSA (negative control)
5mCherry (positive control)

Mowiol was produced due to this protocol: https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf

09/07/16


Fluorescence microscopy 14 (Nikon C2+ confocal microscope): GOPTS + GFP

–> spots with evenly distributed GFP

Fluorescence microscopy 14 (Nikon C2+ confocal microscope):



Spot 1, 3, 4, 5, Positive control

09/14/2016


Sample preparation 16: GOPTS + B53

  1. incubated with B53 (5µl, 60 min)
  2. rinsed and blocked with milk powder(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)
Spot No.Sample
1,2,3GOPTS + B53
4GOPTS + WT (negative control)
5WT (negative control)
6B53 (positive control)
7GOPTS + GFP

09/15/2016


Sample preparation 17: GOPTS + anti-GFP nanobody(Alexa647) + GFP

  1. incubated with nanobody (5µl, 330µg/ml, 60 min)
  2. rinsed and blocked with milk powder(5%) (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 GFP (2µl, 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)
Spot No.Sample
1-4GOPTS + nanobody(Alexa647) + GFP
5GOPTS + milk powder + GFP (negative control)
6GOPTS + milk powder + NB (negative control)
7GOPTS + GFP
8GOPTS (negative control)


Fluorescence microscopy 16 (Nikon C2+ confocal microscope): GOPTS + B53

–> no spores detectable



09/16/16


Fluorescence microscopy 17 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP –> GFP signal: GFP has bound to nanobody


Spot 1-4: NB+GFP







Spot 5 GFP on milkpowder → negative Control - GFP Signal only in glass scratch




Spot 6 NB on MilkPowder → negative Control



Spot 7 GFP on GOPTS → positive control



Spot 8 only GOPTS → negativ control



09/17/16


Sample preparation 18: GOPTS + anti-GFP nanobody + GFP (spotting-mask)

  1. incubation with anti-GFP nanobody ( 2µl/spot, 330µg/ml, 10 min, 4°C)
  2. rinsing and blocking with milk powder(5%) (15 min, 10 rpm)
  3. rinsing with PBS(1x)
  4. incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
  5. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  6. rinsed with ddH2O
  7. (removed mask)
  8. rinsed with ddH2O
  9. dried with compressed air
  10. incubated with Mowiol DABCO (overnight)


09/18/16


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)

  1. incubation with anti-GFP nanobody (2µl/spot, 330µg/ml, 15 min, 4°C)
  2. rinsing with PBS(1x)
  3. (removed mask)
  4. rinsing and blocking with milk powder(5%) (15 min, 2 rpm)
  5. (reattached mask)
  6. incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
  7. (removed mask)
  8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubated with Mowiol DABCO (overnight)


09/19/16


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)

  1. incubation of anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 15 min, 4°C) + B53
  2. incubated on GOPTS-slide (2µl, 15 min)
  3. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  4. rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)

–> problem: no washing away of unbound nanobody


Spot No.Sample
1,2B53 + nanobody(Alexa647) (incubated in solution) on GOPTS
3,4WT + nanobody(Alexa647) (incubated in solution) on GOPTS (negative control)
5,6B53 + nanobody(Alexa647) (incubated in solution) dried on milk powder
7,8WT + nanobody(Alexa647) (incubated in solution) dried on milk powder (negative control)
9,10GOPTS + nanobody(Alexa647) + GFP (positive control)
11,12GOPTS + nanobody(Alexa647) + B53
13,14GOPTS + nanobody(Alexa647) + WT (negative control)
15GOPTS + milk powder + nanobody(Alexa647) (negative control)
16GOPTS + milk powder + B53 (negative control)
17GOPTS + milk powder + WT (negative control)
18GOPTS + GFP (positive control)
19GOPTS + nanobody(Alexa647) (negative control)
20B53 dried (positive control)


09/20/16


Sample preparation 21: anti-GFP nanobody + B53 (in solution)

  1. incubation with anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 30 min, 4°C) + B53
  2. centrifuged (2 min, 13400 rpm)
  3. washed with PBS(1x)
  4. repeated steps 2 and 3
  5. resuspended in PBS(1x) (30µl)
  6. incubated on GOPTS-slide (2µl, 15 min)
  7. rinsed and blocked with milkpowder(5%) (30 min, 10 rpm)
  8. rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)


Spot No.Sample
1B53 + nanobody(Alexa647) (incubated in solution) on GOPTS
2WT + nanobody(Alexa647) (incubated in solution) on GOPTS
3,4B53 + nanobody(Alexa647) dried on milkpowder
5,6WT + nanobody(Alexa647) dried on milkpowder


09/29/16


Sample preparation 22: GOPTS + GFP

  1. incubation with GFP (1 µl, 1.35mg/ml, 30 min)
  2. rinsed and blocked with milk powder(5%) (30 min)
  3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  4. rinsed with ddH2O
  5. dried with compressed air
  6. incubation with Mowiol DABCO (5µl, overnight)
Spot No.Sample
1-5 GOPTS + GFP
6 GOPTS

09/30/16


–> Construct spores are ready for analysis

No.Strain
1151:B. Subtilis CotG(3)
2150 (6)
3159 (7)
4157 (9)

Fluorescence microscopy 22 (Nikon C2+ confocal microscope): GOPTS + GFP

–> spots with evenly distributed GFP

  1. incubation with GFP (1 µl, 1.35mg/ml, 30 min)
  2. spores centrifuged (2 min, 13,400 rpm)
  3. washed with PBS(1x)
  4. step 1 repeated
  5. resuspended in PBS(1x)
  6. incubation on GOPTS-slide(GFP) with spores (4µl, 400,000/µl, incubation time: see table below, 4°C)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed with PBS(1x)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.SampleIncubation time (min)
1GOPTS + GFP + strain 35
2GOPTS + GFP + strain 310
3GOPTS + GFP + strain 315
4GOPTS + GFP + strain 65
5GOPTS + GFP + strain 610
6GOPTS + GFP + strain 615
7GOPTS + GFP + strain 75
8GOPTS + GFP + strain 710
9GOPTS + GFP + strain 715
10GOPTS + GFP + strain 95
11GOPTS + GFP + strain 910
12GOPTS + GFP + strain 915
13, 14GOPTS + GFP (positive control)10
15GOPTS + GFP + WT (negative control)5
16GOPTS + GFP + WT (negative control)10
17GOPTS + GFP + WT (negative control)15
18GOPTS + strain 3 (positive control)10
19GOPTS + strain 6 (positive control)10
20GOPTS + strain 7 (positive control)10
21GOPTS + strain 9 (positive control)10
22GOPTS + WT (negative control)10

10/01/16


Sample preparation 23: GOPTS + GFP + XXX

–> no spores detectable

Sample preparation 24: GOPTS + GFP + XXX (in EtOH resuspended)

  1. incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (10 min, 4°C)
  2. solution centrifuged (4 min, 13,400 rpm)
  3. washed with PBS(1x)
  4. step 2 repeated
  5. resuspended in EtOH (250µl)
  6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed with PBS(1x)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.Sample
1-3GOPTS + GFP + strain 6
4-6GOPTS + GFP + WT
7-9GOPTS + GFP
10-12GOPTS + strain 6
13-15GOPTS + WT

10/02/16


Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + XXX (in EtOH resuspended)

–> EtOH denaturated GFP: very weak signal. No spores detectable

Sample preparation 25: GFP + XXX (in solution)

  1. incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (incubation time: see table below, 4°C)
  2. centrifuged (2 min, 13400 rpm)
  3. washed with PBS(1x)
  4. repeated steps 2 and 3
  5. resuspended in PBS(1x) (20µl)
  6. incubated on GOPTS-slide (4µl, 10 min)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.Sample
1, 2GFP + strain 3
3, 4GFP + strain 6
5, 6GFP + strain 7
7, 8GFP + strain 9
9, 10GFP + WT (negative control)
11, 12GFP + PBS (positive control)
13, 14GFP (positive control)
15, 16WT (negative control)
17strain 3 (negative control)
18strain 6 (negative control)
19strain 7 (negative control)
20strain 9 (negative control)

10/03/16


Sample preparation 26: Incubation test: GFP + XXX (in solution)

  1. incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (incubation time: see tables below, 4°C)
  2. centrifuged (2 min, 13400 rpm)
  3. washed with PBS(1x)
  4. repeated steps 2 and 3)
  5. resuspended in PBS(1x) (20µl))
  6. incubated on GOPTS-slide (4µl, 10 min)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)

Slide 1:

Spot No.SampleIncubation time
1, 2GFP + strain 330 min
3, 4GFP + strain 3(pellet washed with PBS(1x))30 min
5, 6GFP + strain 630 min
7, 8GFP + strain 6(pellet washed with PBS(1x))30 min
9, 10GFP + strain 730 min
11, 12GFP + strain 7(pellet washed with PBS(1x))30 min
13, 14GFP + strain 930 min
15, 16GFP + strain 9 (pellet washed withn PBS(1x))30 min
17, 18GFP + WT (negative control)
19, 20GFP + WT (negative control) (pellet washed with PBS(1x))
21, 22GFP (positive control)
23, 24WT (negative control)
25strain 3 (negative control)
26strain 6 (negative control)
27strain 7 (negative control)
28strain 9 (negative control)

Slide 2:

Spot No.SampleIncubation time
1, 2GFP + strain 360 min
3, 4GFP + strain 3(pellet washed with PBS(1x))60 min
5, 6GFP + strain 660 min
7, 8GFP + strain 6(pellet washed with PBS(1x))60 min
9, 10GFP + strain 760 min
11, 12GFP + strain 7(pellet washed with PBS(1x))60 min
13, 14GFP + strain 960 min
15, 16GFP + strain 9 (pellet washed withn PBS(1x))60 min
17, 18GFP + WT (negative control)
19, 20GFP + WT (negative control) (pellet washed with PBS(1x))
21, 22GFP (positive control)
23, 24WT (negative control)
25strain 3 (negative control)
26strain 6 (negative control)
27strain 7 (negative control)
28strain 9 (negative control)

Slide 3:

Spot No.SampleIncubation time
1, 2GFP + strain 3120 min
3, 4GFP + strain 3(pellet washed with PBS(1x))120 min
5, 6GFP + strain 6120 min
7, 8GFP + strain 6(pellet washed with PBS(1x))120 min
9, 10GFP + strain 7120 min
11, 12GFP + strain 7(pellet washed with PBS(1x))120 min
13, 14GFP + strain 9120 min
15, 16GFP + strain 9 (pellet washed withn PBS(1x))120 min
17, 18GFP + WT (negative control)
19, 20GFP + WT (negative control) (pellet washed with PBS(1x))
21, 22GFP (positive control)
23, 24WT (negative control)
25strain 3 (negative control)
26strain 6 (negative control)
27strain 7 (negative control)
28strain 9 (negative control)

Sample preparation 28: GFP-volume test B: GFP + strain 7 (in solution, washed)

  1. incubation of GFP (volume: see table below, 1,35g/ml) + strain 7 (4µl, 400,000/µl) (90 min, 4°C)
  2. centrifuged (2 min, 13400 rpm)
  3. washed with PBS(1x)
  4. repeated steps 2 and 3)
  5. resuspended in PBS(1x) (20µl)
  6. incubated on GOPTS-slide (2µl, 90 min)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.SampleVolume (GFP)
1, 2GFP + strain 70,5µl
3, 4GFP + strain 71,0µl
5, 6GFP + strain 71,5µl
7, 8GFP + strain 72,0µl
9, 10GFP + strain 72,5µl
11, 12GFP (positive control)4,0µl
13, 14strain 7 (negative control)
15, 16GFP + WT (negative control)0,5µl
17, 18GFP + WT (negative control)1,0µl
19, 20GFP + WT (negative control)1,5µl
21, 22GFP + WT (negative control)2,0µl
23, 24GFP + WT (negative control)2,5µl
25, 26WT (negative control)

Sample preparation 29: Spore-amount test: GFP + strain 7 (in solution)

  1. incubation of GFP (0,5µl, 1,35g/ml) + strain 7 (amount: see table below, 400,000/µl) (90 min, 4°C)
  2. centrifuged (2 min, 13400 rpm)
  3. washed with PBS(1x)
  4. repeated steps 2 and 3)
  5. resuspended in PBS(1x) (20µl)
  6. incubated on GOPTS-slide (2µl, 90 min)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.SampleAmount (spores)
1, 2strain 7 (negative control)25,000,000
3, 4strain 7 (negative control)15,000,000
5, 6strain 7 (negative control)10,000,000
7, 8GFP + strain 725,000,000
9, 10GFP + strain 715,000,000
11, 12GFP + strain 710,000,000
13, 14GFP (positive control)
15, 16WT (negative control)25,000,000
17, 18WT (negative control)15,000,000
19, 20WT(negative control)10,000,000
21, 22GFP + WT (negative control)25,000,000
23, 24GFP + WT (negative control)15,000,000
25, 26GFP + WT (negative control)10,000,000

10/04/16


Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + XXX (in EtOH resuspended)

–>

Fluorescence microscopy 25 (Nikon C2+ confocal microscope): GFP + XXX (in solution)

–>

Fluorescence microscopy 26 (Nikon C2+ confocal microscope): Incubation test: GFP + XXX (in solution)

–>

Fluorescence microscopy 27 (Nikon C2+ confocal microscope): GFP-volume test A: GFP + strain 7 (in solution)

–>

Fluorescence microscopy 28 (Nikon C2+ confocal microscope): GFP-volume test B: GFP + strain 7 (in solution)

–>

Fluorescence microscopy 29 (Nikon C2+ confocal microscope): GFP + strain 7 (in solution)

–>

Sample preparation 30: Wash-test A: GFP + XXX (in solution)

  1. incubation of GFP (1µl, 1,35g/ml) + XXX (25,000.000 spores) (90 min, 4°C)
  2. solution centrifuged (2 min, 13,400 rpm)
  3. washed with PBS(1x)
  4. step 2 and 3 repeated (2x/4x)
  5. resuspended in EtOH (250µl)
  6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed with PBS(1x)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.SampleWashing of GFP-spore solution
1, 2GFP + strain 32x
3, 4GFP + strain 62x
5, 6GFP + strain 72x
7, 8GFP + strain 92x
9, 10GFP + WT (negative control)2x
11, 12GFP + strain 34x
13, 14GFP + strain 64x
15, 16GFP + strain 74x
17, 18GFP + strain 94x
19, 20GFP + WT (negative control)4x
21, 22GFP (positive control)
23WT (negative control)
24strain 3 (negative control)
25strain 6 (negative control)
26strain 7 (negative control)
27strain 9 (negative control)

Sample preparation 31: Wash-test B: GFP + XXX (in solution)

  1. incubation of GFP (1µl, 1,35g/ml) + XXX (25,000.000 spores) (90 min, 4°C)
  2. solution centrifuged (2 min, 13,400 rpm)
  3. washed with TBST(0,005%)
  4. steps 2 and 3 repeated (2x/4x)
  5. resuspended in EtOH (250µl)
  6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed with PBS(1x)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.SampleWashing of GFP-spore solution
1, 2GFP + strain 32x
3, 4GFP + strain 62x
5, 6GFP + strain 72x
7, 8GFP + strain 92x
9, 10GFP + WT (negative control)2x
11, 12GFP + strain 34x
13, 14GFP + strain 64x
15, 16GFP + strain 74x
17, 18GFP + strain 94x
19, 20GFP + WT (negative control)4x
21, 22GFP (positive control)
23WT (negative control)
24strain 3 (negative control)
25strain 6 (negative control)
26strain 7 (negative control)
27strain 9 (negative control)

10/05/16


Fluorescence microscopy 30 (Nikon C2+ confocal microscope): Wash-test A: GFP + XXX (in solution)

–>

Fluorescence microscopy 31 (Nikon C2+ confocal microscope): Wash-test B: GFP + XXX (in solution)

–>

10/06/16


Sample preparation 32: Wash-test C: GFP + spores (in solution)

  1. incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
  2. solution centrifuged (2 min, 13,400 rpm)
  3. washed with TBST(0,005%)
  4. steps 2 and 3 repeated (1x/3x/5x)
  5. resuspended in EtOH (250µl)
  6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
  7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  8. rinsed with PBS(1x)
  9. rinsed with ddH2O
  10. dried with compressed air
  11. incubation with Mowiol DABCO (overnight)
Spot No.SampleWashing of GFP-spore solution (step 3)
1, 2GFP + strain 31x
3, 4GFP + strain 71x
5, 6GFP + WT1x
7, 8GFP + strain 33x
9, 10GFP + strain 73x
11, 12GFP + WT3x
13, 14GFP + strain 35x
15, 16GFP + strain 75x
17, 18GFP + WT5x
19, 20WT (negative control)
21, 22GFP (positive control)

10/07/16


Fluorescence microscopy 32 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (in solution)

–>

Sample preparation 33: Blocking-test A: spores milk powder + GFP (in solution)

  1. solution centrifuged (2 min, 13,400 rpm)
  2. spores washed twice with PBS(1x) (2x 100µl)
  3. spores resuspended and incubated in milk powder(5%) (100µl, incubation time: see table below)
  4. incubation with GFP (5µl, 1,35g/ml, 90 min)
  5. washed with PBS(1x) (3x 100µl)
  6. resuspended in PBS(1x) (20 µl)
  7. incubation on GOPTS-slide (4µl, 30 min, 4°C)
  8. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
  9. rinsed with PBS(1x)
  10. rinsed with ddH2O
  11. dried with compressed air
  12. incubation with Mowiol DABCO (overnight)
Spot No.SampleIncubation time
1, 2strain 3 + milk powder + GFP30 min
3, 4strain 3 + milk powder + GFP60 min
5, 6strain 7 + milk powder + GFP30 min
7, 8strain 7 + milk powder + GFP60 min
9, 10WT + milk powder + GFP (negative control)30 min
11, 12WT + milk powder + GFP(negative control)60 min
13, 14WT + milk powder (negative control)30 min
15, 16WT + milk powder (negative control)60 min
17, 18strain 3 + milk powder (negative control)30 min
19, 20strain 3 + milk powder (negative control)60 min
21, 22strain 7 + milk powder (negative control)30 min
23, 24strain 7 + milk powder (negative control)60 min
25, 26GFP (positive control)30 min

10/08/16


Fluorescence microscopy 33 (Nikon C2+ confocal microscope): Blocking-test A: spores milk powder + GFP (in solution)

–>

10/11/16


Sample preparation 34: Blocking-test B: BSA + GFP (in solution)

  1. solution centrifuged (2 min, 13,400 rpm)
  2. spores (5,000,000) washed with PBS(1x) (3x 100µl)
  3. spores resuspended and incubated in BSA(5%) (100µl, 60 min)
  4. incubation with GFP (50µl, 100µg/ml, 60 min)
  5. washed with TBST(0.05%) (5x 100µl)
  6. resuspended in PBS(1x) (50 µl)
  7. incubation with Mowiol DABCO (overnight)
Spot No.Sample
1, 2strain 3 + BSA + GFP
3, 4strain 7 + BSA + GFP
5, 6WT + BSA + GFP (negative control)
7, 8WT + BSA (negative control)
9, 10strain 3 + milk powder (negative control)
11, 12strain 7 + milk powder (negative control)
13, 14GFP (positive control)

10/12/16


Posted by: iGEM Freiburg

Nanocillus - 'cause spore is more!