Team:Freiburg/NotebookTargeting

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Group 4

Members:Kevin, Christian, Vivi, Nathalie

07/04/2016

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

Nr.Strain
1.B52:B. Subtilis w168 amyE::PcgeA-CotZre-gfp-B0014(CM5)
2.B53:B. Subtilis w168 amyE::PcotYZ-CotZre-gfp-B0014(CM5)
3.B54:B. Subtilis w168 amyE::PcotYZ-CotZin-gfp-B0014(CM5)
4.B55:B. Subtilis w168 amyE::PcotV-CotZre-gfp-B0014(CM5)
5.B56:B. Subtilis w168 amyE::PCotv-CotZin-gfp-B0014(CM5)

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.

07/05/2016

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)

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. 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

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. incubated with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top
Spot numberSample
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

  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 numberSample
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

  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. 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

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 numberSample
1.GOPTS + nanobody + B52
2.GOPTS + nanobody + B53
3.GOPTS + nanobody + B54


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)

07/26/2016

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

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. incubated with Mowiol DABCO (5µl/spot, overnight)

*Positive controls:

  1. Slide with spores B53–> fuorescence observable
  2. B54 –>Fluorescence observable
Spot numberSample
1.GOPTS + GFP (positive control)
2.2 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)


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

07/28/2016

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)

07/29/2016

prepared samples for fluorescence microscopy

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. incubated with Mowiol DABCO (5µl/spot, overnight)
Spot numberSample
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

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

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 numberSample
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

Light microscopy Analyse the slides from 28.7.2016 under light mikroscope

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. incubated with Mowiol DABCO (5µl, overnight)
Spot numberSample
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: 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)

08/15/2016

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)


08/16/2016

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.

Spot numberSampleCondition
1.GOPTS + nanobody + B53washed 2x with PBS(1x) (10 min, 10 rpm)
2.GOPTS + nanobody + B53washed 3x with PBS(1x) (10 min, 10 rpm)
3.GOPTS + nanobody + B53washed 4x with PBS(1x) (10 min, 10 rpm)
4.GOPTS + nanobody + B53washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm)
5.GOPTS + 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 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

–> 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)
Spot numberSample
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.GOPTS + BSA + GFP (negative control)
6.GOPTS + BSA + GFP (negative control)


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 :

GFP auf GOPTSGFP good visible , border clearly visible
Milkprotein on GOPTS+GFPNo GFP observable
BSA on GOPTS + GFPNO GFP observalbe

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.

solutionresult (GFP LASER)
Mowiol/DABCOa vew fluorecent crystals
PBSa vew fluorecent crystals
BSAno fluorescence


08/22/2016

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

08/23/2016

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)
Spot numberSample
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 + - 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)


Spot numberSample
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

  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)


Spot numberSampleCondition
1.GOPTS + nanobody + B53rinsed with PBS(1x)
2.GOPTS + nanobody + B53swivel with PBS(1x) (0.5 min)
3.GOPTS + nanobody + B53washed with PBS(1x) (1 min, 10 rpm)
4.GOPTS + nanobody + B53washed with PBS(1x) (2 min, 10 rpm)
5.GOPTS + nanobody + B53washed with PBS(1x) (3 min, 10 rpm)
6.GOPTS + nanobody + B53washed 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)


06.09.2016

WE coated a slide with mCherry to manage the microscope adjustments.

GFP (slide 4) and mCherry (slide 5) slides were made following the normal protocoll: scheme

slide4

Spotsolutionvolume
1GFP10µl
2GFP10µl
3blocking than GFP5µl
4none0µl

slide5

Spotsolutionvolume
1mCherry10µl
2mCherry10µl
3blocking than mCherry5µl
4none0µl

Mowiol was produced due to this protocol https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf and stored in 1ml aliqouts in the -20 fridge ( only defrost tube twice! ) ; one tube is stored in the normal fridge for instant use (discard in one month )

7.9.16(Kevin)

reproduction of GFP coating with spotting mask:

Spotsolutionvolume
1GFP4 µl
2GFP4µl
3GFP4µl
4GFP4µl
5GFP4µl
6dried GFP4µl

Spot 1, 3, 4, 5, Positive control

14.9.2016(Kevin)

GFP-Spores B53 on GOPTS with spotting mask

Spotsolutionvolume
1B535µl
2B535µl
3B535µl
4WT5µl
5B53 in Moviol4µl
6WT in Moviol4µl

Result: We couldn´t identify any spores with the microscope

15.9.2016(Kevin)

GFP on Nanobody: To get better pictures and have all positiv and negativ control spots on the same slide.

Spot Nrcoatingvolume
1GOPTS+Nanobody 1:10+GFP1µl NB 1µl GFP
2GOPTS+Nanobody 1:10+GFP1µl NB 1µl GFP
3GOPTS+Nanobody 1:10+GFP1µl NB 1µl GFP
4GOPTS+Nanobody 1:10+GFP1µl NB 1µl GFP
5GOPTS+Milk-Blocking+GFP1µl GFP
6GOPTS+Milk-Blocking+NB1µlNB 1:10
7GOPTS+GFP 1µl GFP
8GOPTS-

16.09.

Results GFP on NB Slides from 15.09.

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
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Posted by: iGEM Freiburg

Nanocillus - 'cause spore is more!