Part:BBa_K2114009

aGFPnano_HA_aHelix_cotG

N-terminal fusion of anti-GFP nanobody to spore coat gene cotG by a alpha helical linker.

Usage and Biology

Figure 1: Schematic representation of the resulting fusion protein.

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

This part was used and characterized by the iGEM team Freiburg 2016.

I) Verification of surface localization by flow cytometry

FACS analysis of the surface-displayed fusion construct. Staining of wild type (not transformed) and engineered (transformed with BBa_K2114009) spores with anti-HA antibodies conjugated to Alexa Fluor 647.

The spores of B. subtilis expressing the part BBa_K2114009 were purified by lysozyme treatment to lyse remaining vegetative cells 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 resulting in a slight increase of the fluorescence compared to the stained wild type spores. The low amount of fluorescent spores might be attributible to a low display efficiency using the CotG protein as an anchor.

II) Verification of functionality by binding to GFP

Figure 3: Evaluation of the GFP binding. The spores of B. subtilis expressing the part BBa_K2114001 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_K2114009 exhibited increased fluorescence in comparision to the unmodified wild type spores (Figure 3). The low amount of fluorescent spores might be attributible to a low display efficiency using the CotG protein as an anchor for the anti-GFP nanobody.

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).
3. Radeck, J. et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J. Biol. Eng. 7, 29 (2013).

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]