Part:BBa_K2114009
aGFPnano_HA_aHelix_cotG
N-terminal fusion of anti-GFP nanobody to spore crust gene cotG by a alpha helical linker.
Usage and Biology
This part includes the anti-GFP nanobody (describted by Kubala et al. [Ref]) fused by an alpha helical linker [Ref] to the B. subtilis spore crust gene cotG 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 cotG 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 [REF:BioBrickBox].
Characterization
I) Surface localization
The spores of B. subtilis expressing the part BBa_K2114009 were purified by lysozyme treatment to lyse remaining vegetative cells 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.
II) GFP-binding
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |