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

I) Localizaion: FACS antiHA-Alexa647

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II) Binding of GFP: FACS

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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]