Part:BBa_K4195022

INPNC-ttpA

Biology

INPNC

INPNC is a truncated form of ice nucleation protein (INP) consisting of N- and C- terminal domains (1). It is a membrane protein commonly used to displayed protein on the cell surface (2).

TTPA

TTPA is the phage tail tubular protein A of podophage 7. TTPA can interact with Vp0980, which acts as the receptor of TTPA on the surface of Vibrio parahaemolyticus. TTPA’s binding to Vp0980 mediates phage absorption and subsequent bacterial lysis (3).

Usage and design

Engineering OMVs for treating and preventing AHPND caused by the pathogen V. parahaemolyticus are a significant part of OMEGA project (Operable Magic to Efficiently Getting over AHPND). Based on the efforts of our previous projects in 2020 (AnTea-Glyphosate) and 2021 (SALVAGE), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins was no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), rLvAPN1 (receptor), TTPA (ligand) and TTPB (ligand) (Fig. 1). On one hand, with the receptors displayed, OMVs will gain the function of neutralizing toxins secreted by V. parahaemolyticus. On the other hand, with the assistance of ligands displayed on the surface, OMVs will become a special vector to deliver antimicrobials for the specific pathogen. In summary, we have taken a step closer to the collections of extracellular functional elements (EFE), combining the OMVs, secretion systems and surface display systems which we have been dedicated to since 2020. Learn more information from our Design page.

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.

For this part (INPNC-TTPA), TTPA was fused to the N-terminal of INPNC to surface display for targeting V. parahaemolyticus. Arabinose-inducible system was used in the expression circuit of this basic part at pSB1C3 then the composite part BBa_K4195123 was obtained. We transformed the constructed plasmid into E. coli BL21(DE3) for further verification of its expression and function on the surface of E. coli and OMVs, including the interaction between TTPA and Vp0980.

Characterization

1. Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (3220 bp) can be observed at the position around 3000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195123_pSB1C3.

2. Ability of binding Vp0980 on the surface of engineered bacteria

We used BBa_I0500 promoter and RBS (BBa_B0034) to express INPNC-TTPA protein in E. coli BL21(DE3). The arabinose-induced overnight culture was then incubated with purified Vp0980-his and FITC-labeled anti-His-tag antibody in turn to verify whether the displayed TTPA could bind Vp0980 or not.

Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria (p = 0.0125).

The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Ex = 518 nm) to OD₆₀₀ of positive control (culture was incubated with Vp0980-his) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 3), which indicates that our surface display system works well and the binding ability of TTPA to Vp0980 is retained on the surface of bacteria.

Reference

1. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29, 79-86 (2011).

2. http://2016.igem.org/Team:TJUSLS_China.

3. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerging Microbes Infect. 9, 855-867 (2020).

4. J. L. Valentine et al., Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. Cell Chem. Biol. 23, 655-665 (2016).

5. T. C. Stevenson et al., Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. Proc. Natl. Acad. Sci. U. S. A. 115, E3106-E3115 (2018).

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 330
  Illegal XhoI site found at 927
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 706
  Illegal NgoMIV site found at 964
  Illegal AgeI site found at 1492
  
• 1000
  COMPATIBLE WITH RFC[1000]