Difference between revisions of "Part:BBa K2114002"

Revision as of 20:05, 19 October 2016

aGFPnano_HA_G4S_cotZ

N-terminal fusion of anti-GFP nanobody to spore crust gene cotZ by a flexible GGGGS linker.

Usage and Biology

Figure 1: Schematic representation of the expressed fusion protein.

This part includes the anti-GFP nanobody (describted by Kubala et al. [1]) fused by a flexible GGGGS 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) Verification of surface localizaion by flow cytometry

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This part includes the anti-GFP nanobody[1] fused by a flexible GGGGS 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.

II) Binding of GFP: FACS

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Sequence and Features