Difference between revisions of "Part:BBa K4195006"

Revision as of 09:30, 13 October 2022

his-vp0980

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)

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 Origami 2(DE3). When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (716 bp) can be observed at the position around 750 bp.(Fig. 1)

Fig. 2 The result of regular PCR. Plasmid pET-28a(+).

SDS-PAGE

To extract Vp0980 located on the membrane, 1% Triton X-100 and 1 mmol/L PMSF (both final concentrations) were added to the buffer before ultrasonication, which is a necessary and crucial step for protein purification. As shown in the gel image of his-Vp0980 (Fig. 3), the target protein (20.0 kDa) can be observed at the position around 20 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.

Fig. 3 SDS-PAGE analysis of his-Vp0980 protein. Target bands (20 kDa) can be observed at the position 20kDa.

We can explain that no obvious protein target band in LS because the content of proteins located on the bacterial membrane is minimal.

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