Difference between revisions of "Part:BBa K4195006"

Revision as of 08:25, 12 October 2022

his-vp0980

Biology

Vp0980

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 and design

In order to certify the interaction between Vp0980 and TTPA/TTPB, we construct this part for immunofluorescence and dot blot. We used both BBa_I0500 to construct the expression system and obtained the composite BBa_K4195110, 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.

Fig. 1 Graphic description of the expression gene circuits for display Vp0980 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 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)

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195006_pET-28a(+).

SDS-PAGE

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).

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.

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 11: / Line 11: @@
 ===Usage and design===
-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'' BL21(DE3), 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.
+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.
 [[File:T--XMU-China--his-Vp0980 circuit.png|300px]]
@@ Line 20: / Line 20: @@
 ===Characterization===
-====1. Identification====
+====Identification====
-After transforming the plasmid into <i>E. coli</i> BL21(DE3), colony PCR was used to verify that the plasmid was correct.  Target bands (2218 bp) can be observed at the position around 750 bp (Fig. 2).
+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--BBa K4195006（his-vp0980,colony PCR,BL21(DE3)).png|300px]]
-'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195006_pSB1C3.'''
+'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195006_pET-28a(+).'''
+====SDS-PAGE====
+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).
+'''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.
 ===Reference===