Difference between revisions of "Part:BBa K4195006"

Revision as of 14:42, 12 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

1. 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.

2. 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.

3. 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 ===Biology===
 Vp0980
+<i>V. Parahaemolyticus</i> 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(<i>1</i>).
-''V. parahaemolyticus'' transmembrane protein Vp0980 is predicted to harbour four transmembrane regions, two regions that are inside of the membrane and two regions that 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 <partinfo>BBa_I0500</partinfo> to construct the expression system and obtained the composite part <partinfo>BBa_K4195110</partinfo>, which are assembled on the expression vector pSB1C3 by standard assembly (Fig. 1). The constructed plasmids were transformed into <i>E. coli</i> DH5α and <i>E. coli</i> 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.
-===Usage and design===
+[[File:T--XMU-China-- 110 fig.1.png|400px]]
-In order to certify the interaction between Vp0980 and TTPA/TTPB, we construct this part for immunofluorescence and dot blot. We used both <partinfo>BBa_I0500</partinfo> to construct the expression system and obtained the composite <partinfo>BBa_K4195110</partinfo>, which are assembled on the expression vector pSB1C3 by standard assembly(Fig. 1). The constructed plasmids were transformed into ''E. coli'' SHuffle T7, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing. And we characterize the interaction between Vp0980 and TTPA/TTPB.
+<b>Fig. 1 Gene circuit of <i>vp0980</i> with Histag.</b>
-[[File:T--XMU-China--his-Vp0980 circuit.png|300px]]
+===Characterization===
+====1. 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. 1 Graphic description of the expression gene circuits for display Vp0980 designed in OMEGA project.'''
+[[File:T--XMU-China-- 110fig.2.png|400px]]
+<b>Fig. 2 The result of regular PCR. Plasmid pSB1C3.</b>
-===Characterization===
+====2. SDS-PAGE====
+The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> SHuffle T7. After being cultivated and induced by <i>L</i>-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).
-====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'' Origami2 (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. 2)
+[[File:T--XMU-China--110 fig.3.png|400px]]
-[[File:T--XMU-China-- 191fig.2vpO.png|300px]]
+<b>Fig. 3 SDS-PAGE analysis of protein in lysate of <i>E. coli</i> SHuffle T7 and the elution samples. Target bands (20.0 kDa) can be observed at the position around 20 kDa.</b>
-'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195006_pET-28a(+).'''
+===3. Immoufluorescence===
+The <i>L</i>-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.
-====SDS-PAGE====
+[[File:T--XMU-China--110 fig.4.png|400px]]
-The plasmids verified by sequencing was successfully transformed into ''E. coli'' Origami2 (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 his-Vp0980 (Fig. 2), the bands of target protein (20.0 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).
-[[File:T--XMU-China-- 191fig.vpSDS2.png|400px]]
+<b>Fig. 4 The results of immunofluorescence to verify that Vp0980 is located on the surface of bacteria. (<i>p</i> = 0.0232).</b>
-'''Fig. 3 SDS-PAGE analysis of protein in lysate of ''E. coli'' Origami2 (DE3) and the elution samples.''' Target bands (20.0 kDa) can be observed at the position around 20 kDa.
+The ratio of fluorescence intensity (λ<sub>Ex</sub> = 492 nm, λ<sub>Em</sub> = 528 nm) to OD<sub>600</sub> 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===
-. 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).
+.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. <i>Emerg. Microbes. Infect.</i> <b>9</b>, 855-867 (2020).