Part:BBa_K2114017

aGFPnano_HA_aHelix_cgeA

N-terminal fusion of anti-GFP nanobody to spore crust gene cgeA by an alpha helical linker.

Usage and Biology

Figure 1: Schematic representation of the fusion protein.

This part includes the anti-GFP nanobody [1] fused by an alpha helical linker [2] to the B. subtilis spore crust gene cgeA 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 [3].

Characterization

I) Surface Localization

Figure 2: .

II) GFP binding

Figure 3: .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 681
  
• 1000
  COMPATIBLE WITH RFC[1000]