The E. coli strains were maintained in lysogeny broth medium. Liquid cultures were incubated at 37°C and 250 rpm. Solid cultures were incubated on 1.5 % Agar LB plates. Selection of transformed E. coli was performed by supplementation of the LB medium with the antibiotics ampicillin at a final concentration of 100 µg/mL and chloramphenicol at 25 µg/mL.

Competent E. coli were prepared using the Zymo Research Mix & Go E. coli Transformation Kit according to the protocol of the manufacturer. The E. coli were inoculated and incubated in 50 mL of ZymoBroth™ medium until an OD600 of 0.6 was reached. The cells were harvested by centrifugation and washed with 1x Wash Buffer. After centrifugation the supernatant was removed and the cells were resuspended in pre-cooled 1x Competent Buffer. The competent E. coli were aliquoted into sterile tubes and stored at -80°C until further use. Using the chemically competent E. coli prepared by the Mix & Go Transformation Kit no heat shock or electroporation was necessary.

The competent E. coli were transformed by addition of 2 – 5 µL of a reaction mix containing the appropriate plasmids. E. coli transformed with plasmids harboring an ampicillin resistance were directly spread on agar plates supplemented with ampicilin. For antibiotic resistances the transformed cells were incubated in 300 µL of LB medium at 37°C and 250 rpm prior to spreading on a LB agar plate. The spread cells were incubated over night at 37°C. Subsequently, 5 colonies per plate were picked by a pipet tip, inoculated in 5 mL of LB medium supplemented the appropriate antibiotics and incubated over night at 37°C and 250 rpm.

For storage of bacterial strains 500 µL of the overnight inoculation were added to 500 µL of 30 % glycerol and gently mixed. The stocks were stored at – 80°C for later use.

The preparation of plasmids from transformed E. coli was performed using the kits from Qiagen, PeqLab or Zymo Research according to the protocols of the manufacturers. Prior to the preparation the transformed E. coli were incubated in 5 mL of LB medium supplemented with the appropriate amount of antibiotics. The cells were harvested by centrifugation and lysed at alkaline conditions. The lysate containing the plasmids was loaded on spin columns and eluted in 30 µL of nuclease free water. The concentration of the DNA was determined by a NanoDrop spectrophotometer.

a) Digestion of plasmids for cloning

b) Test digestion

c) Linearization of plasmids for transformation of B. subtilis

Amplified of digested DNA samples were supplemented with the appropriate amount of 10x Orange G loading dye and loaded on a 1% (w/v) agarose TAE gel. Staining of the DNA was performed by the PeqGreen DNA dye at the recommended concentration. The gel was subjected to electrophoresis at 110 V for approximately 30 min or until a sufficient separation of the bands was observable. Visualization of the separated DNA fragments in the gel was performed by an ultraviolet light imager. A 2-log DNA ladder and a 1kb DNA ladder were included as molecular size marker.

Extraction of amplified genes or digested fragments from agarose gels was performed using the QIAquick gel extraction kit from Qiagen according to the protocol of the manufacturer. The bands corresponding to the expected fragment sizes were excised from the gel, lysed with QG buffer at 50°C and loaded on a DNA spin column. The DNA was eluted with 30 µL of nuclease free water and stored at –20°C until further use.

In order to generate the fusion constructs the coat genes, the anti-GFP nanobody and the glutathione S-transferase were amplified by extension PCR to introduce overlapping sequences of at least 25 bp required for Gibson assembly. The iGEM standard backbone pBS1C3 was linearized by treatment with XbaI and SpeI. The assembly was performed with the linearized backbone, an amplified coat gene, and the anti-GFP nanobody or the glutathione S-transferase at molar ratios of 3:1 (insert : vector). The volume of the fragments was adjusted to 2.5 µL with nuclease free water and added to 7.5 µL of prepared 1.33X Gibson master mix. The reaction was incubated for 60 min at 50°C and 2 µL were used to transform chemically competent E. coli DH5α.

a) Colony PCR

b) Extension PCR

c) Thermal cycling

Ligation of amplified B. subtilis coat genes or digested DNA fragments with fusion constructs was performed using the T4 ligase from NEB. The insert, alongside with 50 ng of the linearized backbone were ligated at a molar ratio of 3 : 1 (vector : insert) using 5 units of T4 ligase in 1x T4 reaction buffer in a total reaction volume of 20 µL. The reaction was incubated at room temperature for 30 minutes and 5 µL of the reaction mixture were transformed into chemically competent E. coli DH5α.

For the insertion of small elements into a vector, two oligos containing the desired sequence were mixed at a molar ratio of 1 : 1 and incubated for 15 minutes at 100°C, subsequently the temperature was gradually decreased to room temperature over the course of 3 hours. The annealed oligos were ligated into a plasmid with the appropriate overhangs using the T4 ligase from NEB.

Plasmids that were verified by restriction digestion were sent to sequencing by GATC biotech. The concentration of the DNA samples was adjusted to 30 – 50 ng/µL at a total volume 20 µL. The appropriate primer required for the sequencing reaction were sent in an additional microtube at a concentration of 10 µM. The sequencing results were analyzed using the Geneious software.

The Bacillus subtilis strains were maintained on 1.5 % LB agar. Liquid cultures were grown in LB medium at 37°C and 200 rpm. To germinate Bacillus subtilis spores, the samples were also inoculated in LB medium and incubated overnight.

The cultures were inoculated in LB medium at 37°C and 200 rpm until they reach their exponential growth phase and then resuspended in Difco Sporulation Medium (DSM). After 24 hours of incubation, the cells are sporulated. To purify the samples, they were treated with lysozyme.

To make Bacillus subtilis competent, the samples were inoculated in minimal medium and grow until they reached an OD600 of 1.0-1.3/ml. 600 ng of DNA was added to 400 µl of the competent cells, incubated time temperature shaking and streaked on selective agar.

Approximately 700 Mio spores of Bacillus subtilis were resuspended in lysis buffer containing 50 mM Tris base, 1% SDS, 8 M Urea and 50 mM DTT. The samples were incubated o/n at 60°C while shaking.1 The samples were centrifuged and the SN was taken for further analysis.

40 µl of the spore coat lysates were incubated with 6X Loading dye and boiled for 10 minutes. The prepared spore coat lysates were loaded onto a 10% polyacrylamide gel for SDS-PAGE. The gel was run according to the manufacturer (Bio-Rad). The gel was either stained with Bio-SafeTM Coomassie Stain from Bio-Rad or transferred onto a PVDF membrane for Western Blot analysis. As all constructs contain a HA tag, Anti-HA.11 Epitope Tag Antibody from Biolegend® were used. HRP Goat anti-mouse IgG (minimal x-reactivity) Antibodies from Biolegend® were used for detection. For imaging the Fusion Fx (Vilber Lourmat) was used.

Flow cytometry analysis was performed using 5Mio spores of Bacillus subtilis. HA-Tag (6E2) Mouse mAb (Alexa Fluor® 647 Conjugate) from Cell Signaling Technology® were used for detection of the HA tag. The spores were incubated for 40mins on ice using a dilution of 1:50 in a total volume of 50 µl. Nanobody expressing spores were blocked in 5% BSA for 1h on ice and stained with 100 ug/ml GFP in a total volume of 50µl for 40 mins on ice. All samples were washed 5x with 0.05 % TBS-T and resuspended in 1xPBS for FACS analysis. For imaging the GALLIOS flow cytometer (Beckman-Coulter) was used. The data was analyzed using the FlowJo software.

Plasmid was transformed into Rosetta pLysS competent cells and grown in LB medium supplemented with ampicillin (100 ug/ml) and chloramphenicol (25 ug/ml). Cells were induced at OD600=0.8 w. 1 mM IPTG (end conc.) and incubated overnight at 25°C.

Cells were pelleted at 4000 g 20 min. Resuspended in buffer A, sonicated and purified on HisTrap column.

Questions No. 1: Sample Answer: aGFP nanobody

No. 2: Column Answer: HisTrap FF 5 ml

No. 3: Buffer A Answer: 50 mM Tris 500 mM NaCl 20 mM Imidazole 10 % glycerol pH 8.0

No. 4: Buffer B Answer: 50 mM Tris 150 mM NaCl 400 mM Imidazole 10 % glycerol pH 7.6 The elution buffer was exchanged to PBS using HiPrep 26/10 Desalting column. His tag was cleaved using TEV protease overnight at 4°C. Free his-tag, TEV protease and uncleaved aGFP nanobody were removed from cleaved aGFP nanobody by second pass through HisTrap FF 5 ml column.

The E. coli samples were incubated in 1xPBS and sonified for 10 minutes at 20 %.

The protein concentration was determined via a Lowry assay. 20 µl of the E coli whole cell lysates with wanted protein amount were incubated with 4 µl of 6X Loading dye and boiled for 10 minutes. The prepared lysates were loaded onto a 10% polyacrylamide gel for SDS-PAGE. The gel was run according to the manufacturer (Bio-Rad). The gel was either stained with Bio-SafeTM Coomassie Stain from Bio-Rad or transferred onto a PVDF membrane for Western Blot analysis. As all constructs contain a HA tag, Anti-HA.11 Epitope Tag Antibody from Biolegend® were used. HRP Goat anti-mouse IgG (minimal x-reactivity) Antibodies from Biolegend® were used for detection. For imaging the Fusion Fx (Vilber Lourmat) was used.

References
1. Riesenman, P. J. & Nicholson, W. L. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation // Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Applied and environmental microbiology 66, 620–626 (2000).

Glass slides were washed with EtOH (70 %) and ddH2O and activated by oxygen plasma (300 s, 50 % Power, Plasma Cleaner ZEPTO, Electronic Diener). The activated surface was incubated with GOPTS (50 µl, 2 h, darkness). The slides were then washed with acetone (99,9 %) to remove excess GOPTS.

A spotting mask (silicone, CultureWell™) was used to evenly create sample spots on slides. The spots were incubated with protein solution (GFP/anti-GFP nanobody) (2µl, 15 min, 4°C, darkness) of different concentrations. Blocking was done with a BSA (4%)- or milk protein (5 %)-solution.

A solution of spores (of different concentrations) was pipetted on a nanobody spot and incubated for 20 min. Excess spore were washed off using PBS(1x) (Phosphate Buffered Saline) (10 min, 10 rpm).

After washing the slides were dried with compressed air. A Mowiol/DABCO (2.5 %) mounting solution was used to avoid withering of the proteins (amount depending on size of the cover slip). The samples have been incubated overnight. The coverslips were sealed with nail polish.

Sample analysis was done by fluorescence microscopy (Nikon C2+ confocal).