Part:BBa_K2114002

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

FACS analysis of the surface-displayed fusion construct. Staining of wild type (not transformed) and engineered (transformed with BBa_K2114002) spores with anti-HA antibodies conjugated to Alexa Fluor 647.

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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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_K2114002 exhibited an additional population with increased fluorescence in comparision to the unmodified wild type spores (Figure 3 A).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 447
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]