Part:BBa_K4195021

INPNC-ttpA-his

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-his), a His-tag (6×His) was added to the C-terminal of INPNC-TTPA to verify whether the TTPA is displayed on the surface of engineered bacteria or not. Arabinose-inducible system was used in the expression circuit of this basic part at pSB1C3 then constructed the composite part BBa_K4195122. We transformed the plasmid into E. coli BL21(DE3) for further verification of its location on the surface of E. coli.

Characterization

Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (3238 bp) can be observed at the position around 3000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195122_pSB1C3.

Characterization of displaying cargo proteins

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

Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0024).

The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Em = 518 nm) to OD₆₀₀ of positive control (E. coli harboring BBa_K4195122) is higher than that of negative control (E. coli harboring BBa_K4195007) (Fig. 3), which indicates that our surface display system can successfully display TTPA on the surface of E. coli.

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).

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 484
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 507
  Illegal AgeI site found at 1492
  
• 1000
  COMPATIBLE WITH RFC[1000]