Part:BBa_K4195008

ttpB-his

Biology

TTPB

TTPB is tail tubular protein B of podophage 7. It has been found that TTPB serves as ligands that recognizes the conserved Vibrio receptor Vp0980 to mediate phage adsorption. It binds with Vp0980 of Vibrio parahaemolyticus and then mediates phage adsorption and subsequent bacterial lysis (1).

Usage and design

This circuit was used as the control group to demonstrate surface display of INPNC/ClyA-TTPB and to certify TTPB can interact with the Vp0980 displayed on the surface of E. coli. Both BBa_I0500 were used to construct the expression system and obtained the composite BBa_K4195113, which are assembled on the expression vector pSB1C3 by standard assembly(Fig. 1). The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

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

Characterization

Identification

After the first time of purification, we found that the protein was poorly expressed, so we cloned this part into the expression vector pET-28a(+), then transformed the correct plasmid into E. coli BL21(DE3). When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (2543 bp) can be observed at the position around 3000 bp.(Fig. 2)

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195008_pET-28a(+).

SDS-PAGE

The plasmids verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by IPTG, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of TTPA-his (Fig. 3), the bands of target protein (86.4 kDa) could be observed at the position around 80 kDa on the purified protein lanes (FR).

Fig. 3 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (86.4 kDa) can be observed at the position around 80 kDa.

Proof of the surface display

Immunofluorescence was used to demonstrate the surface display of INPNC/ClyA-TTPB-his. After induced by arabinose, the E. coli was incubated with FITC-labeled anti-His-tag antibody at 37°C for 1h. Microplate reader was used to read the fluorescence intensity (Absorption wavelength: 492 nm, Emission wavelength: 518 nm) and OD₆₀₀. The fluorescence intensity/OD₆₀₀ of INPNC/ClyA-TTPB-his was larger than TTPB-his. The results certified that INPNC/ClyA-TTPB-his was successfully displayed on the outer membrane of E. coli(Fig. 4).

Fig. 4 The results of immunofluorescence to probe the surface display ClyA-TTPB (a) or INPNC-TTPB (b) on the surface of engineered bacteria (p = 0.019 for ClyA-TTPB-his, p = 0.0277 for INPNC-TTPB-his).

The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Em = 518 nm) to OD₆₀₀ of positive control (INPNC/ClyA-TTPB-his) is higher than that of negative control (TTPB-his), which indicates that our surface display system works well.

Reference

1. 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. Emerg. Microbes. Infect. 9, 855-867 (2020).

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 2112
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 765
  Illegal AgeI site found at 1090
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1665