Difference between revisions of "Part:BBa K4195111"

Revision as of 09:15, 12 October 2022

I0500-B0034-ttpA-his-B0015

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). The his-tag (6×His) is added to C-terminal for protein purification.

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 and 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

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 (2266 bp).

Fig. 1 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 ttpA-his tag (Fig. 2), the 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 SHuffle T7 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
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1205
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
Illegal AgeI site found at 1756
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

@@ Line 14: / Line 14: @@
 When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target (2266 bp).
 [[File:T--XMU-China-- 111 fig.2.png|200px]]
 <b>Fig. 1 The result of regular PCR. Plasmid pSB1C3.</b>
 .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 ttpA-his tag (Fig. 2), the target protein (22.4 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).
 [[File:T--XMU-China-- 111SDSfig.2.png|200px]]
 <b>Fig. 2 SDS-PAGE analysis of protein in lysate of <i>E. coli</i> SHuffle T7 and the elution samples. Target bands (22.4 kDa) can be observed at the position around 20 kDa.</b>
+====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 <i>Vibrio parahaemolyticus</i>. <i>Emerging Microbes Infect.</i> <b>9</b>, 855-867 (2020).