Difference between revisions of "Team:Freiburg/NotebookProteins"

(KO/LW/WS) SDS-PAGE, Coomassie staining and Western Blot of B. subtilis whole cell extracts
(1) Preparation of whole cell extracts.
A single B.subtilis (DSM10) colony was picked and inoculated in 5 mL LB-Medium at 250 rpm, 37 °C for 8 hours
The OD600 of the culture was determined by a photometer: 5.34 OD/mL.
Three samples of the inoculation were adjusted to a OD600 of 1.0, 2.0 and 3.0, respectively. The three samples were centrifuged at 13,000 x g for 1 min. The supernatant was discarded and the cell pellet was resuspended in 50 µL of PBS. 10 µL of 6X loading dye were added and subsequently the samples were incubated at 100 °C and 600 rpm for 20 min for cell lysis. Samples 1 and 2 for 40 min.

Four times 50 µL of DSM medium containing spores (see group 3: 04.07.16) were transferred to new tubes and supplemented with 6X LD.
Sample 1: 1X LD
Sample 2: 1X LD, 4% SDS
Sample 3: 1X LD, 0.1M DTT
Sample 4: 1X LD, 4 % SDS, 0.1M DTT
The samples were incubated at 120 °C for 40 min.
The lysed samples from the spores and the cells were centrifuged at 13,000 x g for 1 min and the supernatant was transferred to a new tube. 20 µL of the lysate was loaded on an 10% SDS gel and subjeted to electrophoresis. SDS-PAGE was performed in duplicates and according to protocol. One gel was blotted according to protocol. The blotted membrane was blocked with 5% milk in PBS-Tween(0.01 %). The other Gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

(KO/JP/WS) B.subtilis whole cell extracts
Preparation of whole cell extracts.
Five GFP-displaying-Bacillus colonies (B52-56) were inoculated and grown in 9 ml LB+Cm (diluted 1 to 5000)at 250 rpm, 37 °C for 17 hours

The OD600 of the culture was determined by a photometer: 3.16 OD/mL.6 samples were adjusted to an OD600 of 2.0. The samples were centrifuged at 13,000 x g for 1 min. The supernatant was discarded and the cell pellet was resuspended in 50 µL of H2O with 10 µL of 6X loading dye (sample 1 and 2), buffer A (sample 3 and 4) and buffer B (sample 5 and 6) respectively. The samples were incubated at 99 °C and 900 rpm for 25 min for cell lysis.

Buffer A:
50µL H2O
10 µL LD 6X
6 µL SDS 20%
6.6 µL DTT

Buffer B:
50 µL H2O
10 µL LD 6X
30µL SDS 20%
9 µL DTT

H2O was added to the samples so each had a volume of 99 µL. They were then centrifuged at 13000 x g for 1 min. The supernatant was transferred to a new tube.

(KO/JP/WS)
SDS-PAGE of B. subtilis whole cell extracts
The OD600 of the culture was determined by a photometer: 3.16 OD/mL.6 samples were adjusted to an OD600 of 2.0. The samples were centrifuged at 13,000 x g for 1 min. The supernatant was discarded and the cell pellet was resuspended.

Sample 1 and 2: 1x LD, 2% SDS, 0.1M DTT
Sample 3 and 4: 1x LD, 4% SDS, 0.1M DTT
Sample 5 and 6: 1x LD
The samples were incubated at 99 °C for 25 mins.

The lysed samples from the cells were centrifuged at 13,000 x g for 1 min and the supernatant was transferred to a new tube. 25 µL of the lysate was loaded on a 10% SDS gel and subjected to electrophoresis. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

(KO/LW) SDS-PAGE of B. subtilis whole cell extracts
Bacillus subtilis B43 and B44 were inoculated at 250 rpm, 37 °C, o/n.
The OD600 was determined by a photometer: 6,0/ml
1ml of the samples were centrifuged at 13,000 x g for 1 min, respectively. The supernatant was discarded. The pellets were washed with PBS (samples 1-4) and TBS (samples 5-8). The pellets were resuspended as follows:
samples 1-2: 1ml PBS
samples 3-4: 1ml PBS, 1mM EGTA
samples 5-6: 1ml TBS
samples 7-8: 1ml TBS, 1mM EGTA
The samples were sonified for 10 minutes, with short breaks after 5 minutes, respectively. The samples were centrifuged at 2,000 x g for 4 mins and the supernatant was transferred to a new tube. 10ul 6x LD was added to 50ul of the samples, respectively. The samples were boiled at 99°C for 10 minutes and centrifuged at 13,000 x g for 1 minute. 25ul of the lysate was loaded on an 10% SDS gel and subjected to electrophoresis. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

(KO) Preparation of B. subtilis for whole cell extracts
A single colony of Bacillus subtilis B42 and B43 was picked and inoculated at 250 rpm, 37 °C, o/n, respectively.

(KO) SDS-PAGE of Bacillus subtilis whole cell extracts and spore decoating
The OD600 of the B. subtilis o/n culture was determined by a photometer: 3,1/ml
The OD600 was adjusted to 6/ml. Three samples were taken and centrifuged at 3,000 x g for 2 mins at 24°C. Samples 1 and 2 were washed in either 1xPBS or 1xTBS and resuspended in 500ul of PBS or TBS, respectively. The samples were sonicated for 10 mins, with a short break after 5 mins. The samples were then centrifuged at 3,000 x g for 2 mins at 24°C. 50ul of the supernatant was transferred to a new tube and supplemented with 10ul 6XLD, respectively. The samples were boiled for 10 mins at 99°C at 900rpm.
The pellet of sample 3 was resuspended in 50ul dH2O and 10ul 6xLD. The sample was boiled for 30 mins at 99°C at 900rpm. The sample was centrifuged for 2 mins at 13,000 x g at 25°C. The supernatant was collected for SDS-PAGE.

The OD600 of spores of WT168 B. subtilis was determined by a photometer: 2,1/ml
The OD was adjusted to 10/ml. Two samples were taken and centrifuged for 6 mins at 3,000 x g at 24°C. To Sample 1 1xLD and 4%SDS was added. The sample was boiled at 121°C for 40mins.
To sample 2 1ml of 0,1M NaCl, 0,1M NaOH and 1%SDS was added. The sample was heated at 70°C for 30 mins at 900rpm and centrifuged for 2 mins at 13,000 x g. 50ul of the supernatant was transferred to a new tube and 10ul of 6xLD was added. The sample was boiled for 10 mins at 99°C at 900 rpm.
25ul of the lysates was loaded on a 10% SDS gel and subjected to electrophoresis, respectively. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

(KO) Spore decoating

Sporulation of Bacillus subtilis B53, B55 and B56 was induced (see protocol group 3).
The OD600 of the cultures was determined by a photometer: 3/ml.
1 ml of each sample was taken and centrifuged at 13,000 x g for 3 mins. The samples were resuspended as follows:
sample 1: 50mM Tris-HCl (pH 8,8), 8M Urea, 1%SDS
sample 2: 8M Urea, 1xLD
sample 3: 3M Urea, 1xPBS
Sample 1 was incubated at 37°C for 90mins, sample 2 was incubated at 37°C for 2h and sample 3 was boiled at 95°C for 10mins.
The samples were centrifuged at 13,000 x g for 2 mins. 50ul of the supernatant was incubated with 10ul 6XLD and boiled for 10 mins at 99°C and 7000rpm. The samples were frozen at -20°C o/n.

(KO/LW) SDS-PAGE

25ul of the three samples from the day before were loaded on an 10% SDS gel and subjected to electrophoresis, respectively. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

(WS) [FACS analyis] Analysis of spores and vegetative cells by flow cytometry. Sample preparation: \\Vegetative cells\\: were incubated o/n at 37 °C, 250 rpm. OD600 was photometrically determined at OD600/mL = 5.0. 1 mL was transferred to a tube, centrifuged and washed with PBS. The pellet resuspended with PBS and kept on ice.
\\Spores\\: Aliquoted spores from Danja were thawed and 50 µL per sample were transferred to a new tube, centrifuged and washed with PBS. The pellet was resuspended with PBS and kept on ice.

#1 B.subtilis vegetative cells

#2 B.subtilis W168 spores

#3 B.subtilis W168 spores + vegetative cells

#4 B.subtilis B52 spores

#5 B.subtilis B53 spores

#6 B.subtilis B53 + W168 spores

#7 B.subtilis B54 spores

#8 B.subtilis B55 spores

#9 B.subtilis B56

(KO/DS)
Nanodrop
The sonified samples from group 3 (see Lab book group 3) were taken and the proteinconcentration of the supernatant was measured using the nanodrop at A280:
Bacillus subtilis B46 spores: 0,281 mg/ml
Bacillus subtilis B46 vegetative cells: 1,713 mg/ml
Bacillus subtilis WT168 spores: 0,195 mg/ml
Bacillus subtilis WT168 vegetative cells: 1,812 mg/ml

(KO/LW) B. subtilis spore lysis
3 samples of B53 spores and 3 samples of WT168 spores á 500ul of aliquots with 100 x 10^6spores/500ul were taken and washed with 500ul of PBS once.
The samples were resuspended as follows:
1:
0,1M NaCl
0,1M NaOH
1% SDS
0,1M DTT

2:
50mM Tris base
8M Urea
50mM DTT
1% SDS

3:
3M Urea
1XPBS

All steps were performed for one sample of WT168 and B53 equally.
The samples for 1 were incubated at 70°C for 30 mins, the samples for 2 at 60°C for 90 mins with vortexing every 10mins and the samples for 3 at 99°C for 10mins.
Samples 1 and 2 showed colour change (red) –> DSM needs to be washed out better
The supernatants of each sample were transferred to a new tube, respectively.
Because of the colour change, a measurement with nanodrop was not possible.
The pellets of sample 2 were visibly smaller than the pellets of 1 and 3.

(KO/LW) B. subtilis spore lysis
Aliquoted and purified spores from WT 168 and B54 (see Group 3) were taken.
The samples were resuspended as follows:
1:
0,1M NaCl
0,1M NaOH
1% SDS
0,1M DTT

2:
50mM Tris base
8M Urea
50mM DTT
1% SDS

All steps were performed for one sample of WT168 and B53 equally.
The samples for 1 were incubated at 70°C for 30 mins and the samples for 2 at 60°C for 90 mins with vortexing every 10mins.
Red colouring was still visible.
The samples were centrifuged at 13,000 x g for 1,5 mins. Supernatants were transferred to new tubes and stored at -20°C o/n.

10ul 6xLD was added to 50ul of the before frozen samples, respectively. The samples were boiled at 99°C for 10 minutes. 25ul of the lysate was loaded on a 10% SDS gel and subjected to electrophoresis. As controls, vegetative cells of WT168 were treated with lysozyme and purified (see group 3) and spores of WT168 were treated with lysozyme and washed (group 3). SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

FACS (WS/KO)
1Mio spores of Bacillus subtilisWT168, B53 and B54 were washed with 1xPBS and then incubated in 500 µL of 1XPBS, 2. aGFP-nanobody conj. w Alexa647 (0,15mg/ml)(1:500) and 3. aGFP-nanobody conj. w Alexa647 (1:1000) for 40mins at 4°C.
The samples were washed 3x with 1xPBS (11,000 x g for 2mins) and then incubated in 500ul of PBS for FACS analysis.
08.08.2016 Alexananobody.pdf

Spore decoating (KO/VJ)
Two aliquots of 500 Mio spores of WT168 and B56 were treated with lysozyme according to protocol (group 3). The pellets were washed 6 times in 1xPBS and resuspended in 1xPBS for heat shock at 65°C for 1h.
The purified spores were incubated as follows:
1. (each for 1 sample WT168, 1 sample B56 respectivley):
0,1M NaCl
0,1M NaOH
1% SDS
1uM DTT
2.:
50mM Tris base
8M Urea
1uM DTT
1% SDS
The samples were incubated at 30°C at 450rpm o/n for SDS-PAGE analysis.

SDS-PAGE of spore lysates (KO)
Samples from the day before were centrifuged and the SN was transferred to a new tube. Two samples of vegetative cells were treated with lysozyme, one sample was washed with PBS six times. The other sample was centrifuged and the SN transferred to a new tube. Purified and washed spores of WT168 (group3) were centrifuged and the SN was transferred to a new tube. 50ul of all SNs were taken and 10ul of 6xLD was added. The samples were boiled at 99°C for 10 mins. 25ul of the lysate was loaded on a 10% SDS gel and subjected to electrophoresis. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

FACS (WS/KO)
10Mio spores of WT168, B53 and B54 (purified and washed from group3) were incubated in 500ul of 1XPBS respectively, 1. was left untreated, 2. incubated in aGFP-nanobody conj. w Alexa647 (0,15mg/ml)(1:5,000) and 3. incubated in aGFP-nanobody conj. w Alexa647 (1:10,000) for 40mins at 4°C. The samples were washed 5x in 0,05% TBS-T and resuspended in 1ml PBS for FACS analysis.
14.08.2016 Alexananobody.pdf
weight of pellet in eppendorfer tubes before and after lysis:

Spore decoating (KO/LW)
Two aliquots of 500 Mio spores of WT168 and B52 were treated with lysozyme according to protocol (group 3). The pellets were washed 6 times in 1xPBS.
The purified spores were incubated as follows:
1. (each for 1 sample WT168, 1 sample B52 respectively):
0,1M NaCl
0,1M NaOH
1% SDS
1uM DTT
2.:
50mM Tris base
8M Urea
1uM DTT
1% SDS
The samples were incubated at 30°C at 450rpm o/n for SDS-PAGE analysis.
One additional sample of B52 was treated with 2 and incubated at 70°C o/n.

Samples from the day before were centrifuged and the SN was transferred to a new tube. Two samples of vegetative cells were treated with lysozyme, one sample was washed with PBS six times. The other sample was centrifuged and the SN transferred to a new tube. Purified and washed spores of WT168 (group3) were centrifuged and the SN was transferred to a new tube. 50ul of all SNs were taken and 10ul of 6xLD was added. The samples were boiled at 99°C for 10 mins. 25ul of the lysate was loaded on a 10% SDS gel and subjected to electrophoresis. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

Cell lysis (KO)
Spores of B. subtilis WT168 and B46 were treated with lysozyme (1:6) for 1h and washed with PBS 6x.
They were incubated as follows:
0,1M NaCl
0,1M NaOH
1% SDS
1uM DTT
2.:
50mM Tris base
8M Urea
1uM DTT
1% SDS
The samples were incubated at 30°C at 450rpm o/n for SDS-PAGE analysis.

The samples from the day before were centrifuged, the SN was transferred to a new tube and frozen.
Western Blot
TALEN HA tagged protein lysates were prepared for SDS-PAGE by boiling for 10mins at 99°C with 1xLD.
Two gels for SDS-PAGE were loaded with HA tagged protein lysates, respectively:
lane 1:marker
lane 2: 1ug
lane 3: 10ug
lane 4: 20ug
lane 5: 30ug
lane 6:marker
lane 7: 1ug
lane 8: 10ug
lane 9: 20ug
lane 10: 30ug
After SDS-PAGE the gel was taken and the proteins were transferred to a membrane according to protocol, for each gel respectively.
The membranes were blocked with 5% milk TBS-T at 4°C o/n.

The membranes from the day before were cut in half.
Primary antibodies aHA, mouse were added to gel 1, 2 and 3 in the dilutions (in 5ml 5% Milk TBS-T (0,1%) respectively):
A: 1:1000
B: 1:2500
C: 1:5000
To membrane D 15ml aHA, rabbit 1:1000 in 5% milk TBS-T were added.
The membranes were incubated for 3h at RT. They were washed in TBS-T for 15min at RT and incubated in secondary antibody for 45min at RT:
A,B and C: 1:5000 aMouse in 5ml 5% milk TBS-T
D: 1:5000 aRabbit in 15ml 5% milk TBS-T
Membranes were washed 3x for 15min at RT in TBS-T. 1ml of Peroxide solution and 1ml of Luminol/Enhancers were added and chemiluminescence was measured in the fusion.

FACS
Samples from group 3 were taken for FACS analysis.
08.08.2016 Alexananobody.pdf

SDS-PAGE (KO)
50ul of the frozen samples from 20.8. and 17.8. were transferred to a new tube and 10ul 6xLD was added, respectively. The samples were boiled at 99°C for 10 minutes. 25ul of the lysate was loaded on an 10% SDS gel and subjected to electrophoresis. SDS-PAGE was performed according to protocol. The gel was coomassie-stained with Bio-Safe Coomassie Stain from Bio-Rad according to the protocol of the manufacturer.

Transformation of E coli (NV)
E. coli BL21 were transformed with pIG16_23(pET303-aGFPnano_TEV_10His). The culture was spread on a LB+Amp plate and inoculated o/n.

Transformation of e coli (KO)
A single colony of the plate from the day before was picked nd inoculated in 10ml of LB+Ampicillin o/n at 37°C and 200rpm.

The culture from the day before as diluted 1:100 in 12ml of LB+Amp and inoculated until the OD600 was 0,63/ml. 10 ml of the culture was incubated with 0,4mM IPTG and inoculated for 6h at 30°C and 200rpm.
The samples were centrifuged at 6,000 x g for 15 mins and incubated in 1ml of 1xPBS. The cells were sonified for 10mins and centrifuged at 13,000 x g for 5 mins. The supernatant was used for protein purification using the His-Spin Protein Miniprep™ from ZymoResearch. The purification was performed according to protocol

Transformation of E coli
A single colony of the plate from the day before was picked and inoculated in 10ml of LB+Ampicillin for o/n at 37°C and 200rpm.

Transformation of E coli
The culture from the day before as diluted 1:100 in 12ml of LB+Amp and inoculated until the OD600 was 0,63/ml. 10 ml of the culture was incubated with 0,4mM IPTG and inoculated for 24h at 30°C and 200rpm.

Protein purification
The samples were centrifuged at 6,000 x g for 15 mins and incubated in 1ml of 1xPBS. The cells were sonified for 10mins and centrifuged at 13,000 x g for 5 mins. The supernatant was used for protein purification using the His-Spin Protein Miniprep™ from ZymoResearch. The purification was performed according to protocol.

SDS-PAGE of purified nanobody
2,4ug of the purified nanobody was transferred to a new tube and 6xLD was added. The samples were boiled at 99°C for 10 minutes. As controls, TALEN whole cell lysates at the concentrations 1ug and 5ug were used. 24ul of the lysate was loaded on a 10% SDS gel and subjected to electrophoresis. SDS-PAGE was performed according to protocol. The gel was Coomassie-stained with Bio-Safe Coomassie solution from Bio-Rad according to the protocol of the manufacturer.

preparation of E. coli whole cell lysates
Two liquid cultures of E. coli transformed with constructs 143 and 149 were centrifuged and resuspended in 2ml PBS. The samples were sonified for 10mins and centrifuged at 13,000 g at 4°C for 5mins. The supernatant was transferred to a new tube. The protein concentrations were measured performing a Lowry assay acoording to protocol.
E coli with 143: 1,3 ug/ul
E coli with 149: 0,8 ug/ul

17ug , 15ug, 10ug and 5ug were loaded onto a 10% SDS-gel and subjected to electrophoresis, respectively. SDS-PAGE was performed according to protocol. The gel was blotted according to protocol. The blotted membrane was blocked with 5% milk in TBS-Tween (0.1%) over-night.

Western Blot of E. coli lysates
The blocked membrane was washed with TBS-T for 15mins and then incubated in primary antibody aHA, mouse solution (1:2500 in 5ml of 5%milk TBS-T). The membrane was incubated for 3h at RT. It was washed in TBS-T for 15min at RT and incubated in secondary antibody for 45min at RT: 1:5000 aMouse in 5ml 5% milk TBS-T
Membranes were washed 3x for 15min at RT in TBS-T. 1ml of Peroxide solution and 1ml of Luminol/Enhancers were added and chemiluminescence was measured in the fusion fx.

preparation of E. coli whole cell lysates
One liquid culture of E. coli DH5a and five liquid cultures of E. coli transformed with constructs 131, 133, 134, 137 and 138 were centrifuged and resuspended in 1ml PBS. The samples were sonified for 10mins and centrifuged at 13,000 g at 4°C for 5mins. The supernatant was transferred to a new tube. The protein concentrations were measured performing a Lowry assay acoording to protocol.
E coli with 131: 5,3 ug/ul
E coli with 133: 5,4 ug/ul
E coli with 134: 4,6 ug/ul
E coli with 137: 5,1 ug/ul
E coli with 138: 5,9 ug/ul
E coli DH5a: 1,9 ug/ul
The lysates were stored at -80 degrees.

SDS-PAGE and Western Blot
The lysates of DH5a, 131, 133, 134, 137, 138, 143 and 149 were loaded onto the first 10% SDS-gel at a concentration of 17ug and subjeted to electrophoresis, respectively.
The lysates of DH5a, 131, 133, 134, 137, 138 and 143 at a concentration of 25ug and 149 at a concentration of 17ug were loaded onto the second 10% SDS-gel and subjeted to electrophoresis, respectively.
SDS-PAGE was performed according to protocol. The gels were blotted according to protocol. The blotted membranes were blocked with 5% milk in TBS-Tween (0.1%) over-night.

Western Blot of E. coli lysates
The blocked membranes were washed with TBS-T for 15mins and then incubated in primary antibody aHA, mouse solution (1:2500 in 5ml of 5%milk TBS-T). The membranes were incubated for 3h at RT. They were washed in TBS-T for 15min at RT and incubated in secondary antibody for 45min at RT: 1:5000 aMouse in 5ml 5% milk TBS-T
Membranes were washed 3x for 15min at RT in TBS-T. 1ml of Peroxide solution and 1ml of Luminol/Enhancers were added and chemiluminescence was measured in the fusion fx.

Nanobody expression
A single colony of E coli transformed w pIG16_23 was picked and inoculated in 10ml LB+Amp o/n.

DotBlot
The spores of B. subtilis 150, 151, 157, 159, 160, each in WT and respective knock-out, WT, dCotG, dCgeA, dCotZ were dot-blotted at concentrations of 2Mio, 200,000 and 20,000. As positive controls 10ug and 15ug of 137 and 8ug and 12ug of TALEN lysate were dot-blotted. The membrne was left to dry for 2h at RT. After drying it was stained in Ponceau S staining solution for 45mins at RT. The membrane was destained and blocked o/n at 4°C.

Spore decoating
500Mio spores of B. subtilis 160, 159, 157, 151, 150, Wildtype, dCotG, dCotZ and dCgeA were incubated in:
50mM Tris base
8M Urea
1uM DTT
1% SDS
They were incubated o/n at 750rpm and and 60°C.

FACS of spores
10Mio spores of B. subtilis 160, 150, 159 and Wildtype were treated as follows:
1. incubation in aHA antibody Alexa647 1:50(total volume: 50ul) for 2mins on ice
2. incubation in aHA antibody Alexa647 1:50(total volume: 50ul) for 10mins on ice
all except for 160 were additionaly treated with:
3. incubation in GFP 1ug/ml (total volume: 50ul) for 2mins on ice
4. incubation in GFP 1ug/ml (total volume: 50ul) for 10mins on ice
As controls one sample of each was left untreated.

Nanobody expression
The E. coli culture from the day before was diluted 1:100 and let grown to an OD600 of 0,8/ml. The culture was induced with 1mM IPTG at 30°C o/n.

FACS of spores
5Mio spores of B. subtilis 160, 159, 157, 151, 150 and Wildtype were treated as follows:
1. incubation in aHA antibody Alexa647 1:50(total volume: 50ul) for 20mins on ice
2. incubation in aHA antibody Alexa647 1:50(total volume: 50ul) for 40mins on ice
all except for 160 were additionaly treated with:
3. incubation in GFP 1ug/ml (total volume: 50ul) for 20mins on ice
4. incubation in GFP 1ug/ml (total volume: 50ul) for 40mins on ice
As controls one sample of each was left untreated.

Western Blot of decoated spores
The samples from 2.10. were taken for SDS-PAGE analysis and Western Blot. SDS-PAGE and Western Blot were performed according to protocol. After blotting the membrane was stained with Ponceau S staining. The membrane was destained in water and blocked in 5% milk TBS-T o/n at 4°C.

Nanobody expression
The E. coli sample from the day before were sonified for 10mins at 20%. The sample was centrifuged at 13,000 x g for 5mins t 4°C. The lysate and the pellet were taken for SDS-PAGE. SDS-PAGE was performed according to protocol. The gel was Coomassie-stained with Bio-Safe Coomassie solution from Bio-Rad according to the protocol of the manufacturer.

E. coli expressing 131, 134 and 138
E. coli 131, 134 and 138 were taken from glycerol stocks and inoculated o/n.

E. coli expressing 131, 134 and 138
The E. coli cultures from the day before were diluted 1:100 and let grown to an OD600 of 0,8/ml. The culture was induced with 1mM IPTG at 25°C o/n.

E. coli expressing pIG16_23
The E. coli culture from the day before was diluted 1:100 and let grown to an OD600 of 0,8/ml. The culture was induced with 1mM IPTG at 30°C o/n.

Western Blot of spores
The blocked membranes were washed with TBS-T for 15mins and then incubated in primary antibody aHA, mouse solution (1:2500 in 5ml of 5%milk TBS-T). The membranes were incubated for 3h at RT. They were washed in TBS-T for 15min at RT and incubated in secondary antibody for 45min at RT: 1:5000 aMouse in 5ml 5% milk TBS-T
Membranes were washed 3x for 15min at RT in TBS-T. 1ml of Peroxide solution and 1ml of Luminol/Enhancers were added and chemiluminescence was measured in the fusion fx.

FACS of spores
5Mio spores of B. subtilis 159, 157, 150 and Wildtype were treated as follows:
1. incubation in aHA antibody Alexa647 1:50(total volume: 50ul) for 40mins on ice
2. incubation in GFP 10 ug/ml (total volume: 50 ul) for 40mins on ice
4. incubation in GFP 100 ug/ml (total volume: 50 ul) for 40mins on ice

Spore decoating
700Mio spores of B. subtilis 160, 159, 157, 151, 150, WT, dCotG, dCotZ and dCgeA were incubated in 100ul of:
50mM Tris base
8M Urea
1uM DTT
1% SDS
They were incubated o/n at 750rpm and and 60°C.

Western Blot of decoated spores
The samples from 5.10. were taken for SDS-PAGE analysis and Western Blot. SDS-PAGE was performed in duplicates and according to protocol. One gel was Coomassie-stained with Bio-Safe Coomassie solution from Bio-Rad according to the protocol of the manufacturer. The other gel was blotted according to protocol. After blotting the membrane was stained with Ponceau S staining. The membrane was destained in water and blocked in 5% milk TBS-T o/n at 4°C.

E. coli expressing 131, 134 and 138
The E. coli samples from the day before were sonified for 10mins at 20%. The samples were centrifuged at 13,000 x g for 5mins at 4°C. A Lowry assay was performed according to protocol. The samples were aliquoted and stored at -80°C.

Nanobody expression
The E. coli sample from the day before were sonified for 10mins at 20%. The sample was centrifuged at 13,000 x g for 5mins at 4°C. The lysate and the pellet were frozen at -20°C overnight.

Western Blot of spores
The blocked membranes were washed with TBS-T for 15mins and then incubated in primary antibody aHA, mouse solution (1:2500 in 5ml of 5%milk TBS-T). The membranes were incubated for 3h at RT. They were washed in TBS-T for 15min at RT and incubated in secondary antibody for 45min at RT: 1:5000 aMouse in 5ml 5% milk TBS-T
Membranes were washed 3x for 15min at RT in TBS-T. 1ml of Peroxide solution and 1ml of Luminol/Enhancers were added and chemiluminescence was measured in the fusion fx.

Nanobody expression
The frozen lysates from the day before were taken and SDS-PAGE was performed according to protocol. The gel was Coomassie-stained with Bio-Safe Coomassie solution from Bio-Rad according to the protocol of the manufacturer

FACS of spores
5Mio spores of B. subtilis 159, 157 and Wildtype were treated as follows:
1. incubation in aHA antibody Alexa647 1:50(total volume: 50ul) for 40mins on ice
2. blocking in 5% BSA (50 ul) for 1h on ice, washing 3x with 1xPBS, incubation in GFP 100 ug/ml (total volume: 50 ul) for 40mins on ice
4. blocking in 5% BSA (50 ul) for 1h on ice, incubation in BSA and GFP 100 ug/ml (total volume: 50 ul) for 40mins on ice

FACS of spores
5Mio spores of B. subtilis 152, 153, 156, 167, 168 and Wildtype were incubated in aHA antibody Alexa647 1:50(total volume: 50ul) for 40mins on ice