Difference between revisions of "Part:BBa K2114001"

Revision as of 22:17, 18 October 2016

aGFPnano_HA_aHelix_cotZ

N-terminal fusion of anti-GFP nanobody to spore crust gene cotZ.

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 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. [Figure of construct: Representation of the fusion protein.]

Characterization

I) Expression: WB
II) Localizaion: FACS antiHA-Alexa647
III) Binding of GFP: FACS

References

1. Kubala et al. 2. Surface display alpha helical linker Sequence and Features