Part:BBa_K2114001
aGFPnano_HA_aHelix_cotZ
N-terminal fusion of anti-GFP nanobody to spore coat gene cotZ by an alpha helical linker.
Usage and Biology
This part includes the anti-GFP nanobody 1 fused by an alpha helical linker 2 to the B. subtilis spore crust protein CotZ in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotZ gene was amplified from the genome of B. subtilis and the anti-GFP nanobody was amplified from an expression plasmid. The HA tag and the alpha helical linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly.
Characterization
This part was used and characterized by the iGEM team Freiburg 2016.
I) Expression analysis by Western blotting
BBa_K2114001 was assembled into pBS1C 3 alongside with the PCotYZ-RBS promoter (BBa_K2114000) and transformed into competent B. subtilis. Sporulation was induced by starvation in minimal medium. The spore coat proteins were extracted and analysed by SDS-PAGE and Western blotting.
BBa_K2114001 was cloned alongside with The promoter PCotYZ-RBS (BBa_K2114000) into the integration vector pBS1C by 3A assembly. After transformation the cells were selected by chloramphenicol resistance and screened for the disruption of the amyE gene on starch agar plates. Subsequently the positive clones were further cultivated and sporulation was induced by nutrient starvation. The resulting spores were purified from vegetative cells with lysozyme and analyzed by SDS-PAGE and Western blotting. The immunostaining with anti-HA antibodies resulted in the visualization of the expected band at approximately 33 kDa. Additional bands at higher molecular weight were hypothesized to be results from the high cross-linking of spore coat proteins responsible for the enormous rigidity and stability of the spores 4.
II) Verification of surface localizaion by immunostaining
The spores of B. subtilis expressing the part BBa_K2114001 were purified by lysozyme treatment and stained with anti-HA antibodies conjugated to Alexa Fluor® 647 (Cell Signaling Technology®). The antibody could only access surface-localized HA epitopes of the expressed fusion genes and could confirm the successful display of the heterologous protein on the surface of the modified spores while the wild type spores did not exhibit any increase in the fluorescence.
III)Verification of functionality by binding to GFP
To verify the functionality of the expressed fusion construct containing the anti-GFP nanobody the spores were incubated with purified GFP and analyzed by flow cytometry in order to detect the fluorescence. Spore expressing the the part BBa_K2114001 exhibited increased fluorescence in comparision to the unmodified wild type spores.
References
1. Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 19, 2389–2401 (2010).
2. Hinc, K., Iwanicki, A. & Obuchowski, M. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb. Cell Fact. 12, 22 (2013).
3. Radeck, J. et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J. Biol. Eng. 7, 29 (2013).
4. Driks, A. Bacillus subtilis Spore Coat. Microbiol. Mol. Biol. Rev. 63, 1–20 (1999).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 465
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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