Difference between revisions of "Part:BBa K4195007"

Latest revision as of 08:52, 12 October 2022

ttpA-his

Biology

TTPA is the phage tail tubular protein A of podophage 7. TTPA can interact with Vp0980, which acts as the receptor of TTPA on the surface of Vibrio parahaemolyticus. TTPA’s binding to Vp0980 mediates phage absorption and subsequent bacterial lysis (1).

Usage and design

A His-Tag (6×His) was added to the C-terminal of TTPA for purifying this protein. Arabinose-inducible system was used in the expression circuit at pSB1C3 then composite part BBa_K4195111 was obtained. We transformed the plasmid into E. coli SHuffle T7 to obtain the protein for further biochemical characterizations.

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 (764 bp) can be observed at the position around 750 bp.(Fig. 1)

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195007_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. 2), the bands of target protein (22.4 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (22.4 kDa) can be observed at the position around 20 kDa.

Western blot

This year, we shared deep collaborations with a first-year team, CUG-China. With the help of them for offering the operation of western blot, we could confirm that the TTPA-his was expressed indeed (Fig. 3), after struggling to obtain the purified protein for several rounds mentioned above.

Fig. 3 Western blot analysis (anti-His-tag) of protein in lysate supernatant of E. coli SHuffle T7. Target bands (22.4 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. Emerging 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
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 520
1000
COMPATIBLE WITH RFC[1000]

@@ Line 9: / Line 9: @@
 ===Characterization===
 ====Identification====
-When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (764 bp) can be observed at the position around 750 bp (Fig. 1).
+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 (764 bp) can be observed at the position around 750 bp.(Fig. 1)
 [[File:T--XMU-China-BBa_K4195007.png|200px]]
-'''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195007_pSB1C3.'''
+'''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195007_pET-28a(+).'''
-====Purification and proof of expression====
+====SDS-PAGE====
-We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express TTPA-his protein in ''E. coli'' SHuffle T7. GE AKTA Prime Plus FPLC system was implemented to get purified TTPA protein. Target bands (22.4 kDa) can be observed at the position around 20 kDa(Fig. 2).
+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. 2), the bands of target protein (22.4 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).
 [[File:T--XMU-China--BBa_K4195007(SDS-PAGE).png|300px]]
-'''Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples.'''
+'''Fig. 2 SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21(DE3) and the elution samples.''' Target bands (22.4 kDa) can be observed at the position around 20 kDa.
+====Western blot====
+This year, we shared deep collaborations with a first-year team, CUG-China. With the help of them for offering the operation of western blot, we could confirm that the TTPA-his was expressed indeed (Fig. 3), after struggling to obtain the purified protein for several rounds mentioned above.
+[[File:T--XMU-China-- ｔｔｐａｗｂ.png|200px]]
+'''Fig. 3 Western blot analysis (anti-His-tag) of protein in lysate supernatant of ''E. coli'' SHuffle T7.''' Target bands (22.4 kDa) can be observed at the position around 20 kDa.
 ====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. Emerging Microbes Infect.'' '''9''', 855-867 (2020).