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.

The spores of B. subtilis expressing the part BBa_K2114002 were purified by lysozyme treatment and stained with anti-HA antibodies conjugated to Alexa Fluor® 647 (Cell Signaling Technology®). The antibody could only access surface-localized HA epitopes of the expressed fusion genes and could confirm the successful display of the heterologous protein on the surface of the modified spores while the wild type spores did not exhibit any increase in the fluorescence.

II) Verification of functionality by binding to GFP

Figure 3: Evaluation of the GFP binding. The spores of B. subtilis expressing the part BBa_K2114002 and wild type spores were incubated with purified GFP and analyzed by flow cytometry.(A) The scatter plots indicates the increase of fluorescence of the spores expressing and displaying the part BBa_K2114002. (B) Depiction of the fluorescence intensity as histogram illustrates the difference in the mean fluorescence intensity between wild type and modified spores.

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. Spores expressing the part BBa_K2114002 exhibited increased fluorescence in comparison to the unmodified wild type spores (Figure 3).

References

1. Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 19, 2389–2401 (2010).
2. Hinc, K., Iwanicki, A. & Obuchowski, M. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb. Cell Fact. 12, 22 (2013).
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]