Difference between revisions of "Part:BBa K4195110"

Revision as of 09:38, 12 October 2022

I0500-B0034-his-vp0980-B0015

Biology

Vp0980 V. Parahaemolyticus transmembrane protein Vp0980 is predicted to harbour four transmembrane regions. Two regions are inside of the membrane and two regions are outside of the membrane. The regions outside of the membrane are likely to specially binds phage tail tubular proteins TTPA and TTPB to mediate phage adsorption(1).

Usage

In order to certify the interaction between Vp0980 and TTPA/TTPB, we construct this part for immunofluorescence and dot blot. We used BBa_I0500 to construct the expression system and obtained the composite part BBa_K4195110, which are assembled on the expression vector pSB1C3 by standard assembly (Fig. 1). The constructed plasmids were transformed into E. coli DH5α and E. coli SHuffle T7, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing. And we characterized the interaction between Vp0980 and TTPA/TTPB.

Fig. 1 Gene circuit of vp0980 with Histag.

Characterization

Agarose gel electrophoresis (AGE)

When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target (2218 bp).

Fig. 2 The result of regular PCR. Plasmid pSB1C3.

SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli SHuffle T7. After being cultivated and induced by L-arabinose, 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 his-Vp0980 (Fig. 3), the target protein (20.0 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).

Fig. 3 SDS-PAGE analysis of protein in lysate of E. coli SHuffle T7 and the elution samples. Target bands (20.0 kDa) can be observed at the position around 20 kDa.

Immoufluorescence

The L-arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody to test whether the Vp0980 located on the surface of engineered bacteria or not.

Fig. 4 The results of immunofluorescence to verify that Vp0980 is located on the surface of bacteria. (p = 0.0232).

The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Em = 528 nm) to OD₆₀₀ of positive control (bacteria with expression of his-Vp0980) is higher than that of negative control (bacteria without expression of his-Vp0980) (Fig. 4), which indicates that Vp0980 is located on the surface of bacteria.

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