Difference between revisions of "Part:BBa K4195089"
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===Characterization===
 
===Characterization===
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1. Agarose Gel Electrophoresis (AGE)
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====1. Agarose Gel Electrophoresis (AGE)====
 
After transforming the plasmid into <i>E. coli</i> BL21(DE3), colony PCR was used to verify that the plasmid was correct. The expected result was obtained (764 bp).
 
After transforming the plasmid into <i>E. coli</i> BL21(DE3), colony PCR was used to verify that the plasmid was correct. The expected result was obtained (764 bp).
  
 
[[File:T--XMU-China--089 fig.1.png|400px|center]]
 
[[File:T--XMU-China--089 fig.1.png|400px|center]]
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<p align="center"><b>Fig. 1 The result of colony PCR.</b>
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<p align="center"><b>Fig. 1 The result of colony PCR.</b></p>
  
  
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====2.SDS-PAGE====
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2.SDS-PAGE
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The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> 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-myc tag (Fig. 2), the target protein (23.6 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).
 
The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> 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-myc tag (Fig. 2), the target protein (23.6 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).
  
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[[File:T--XMU-China--4195089 fig.2 .png|400px|center]]
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[[File:T--XMU-China--4195089 fig.2 .png|600px|center]]
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<p align="center">><b>Fig. 2 SDS-PAGE analysis of protein in lysate of <i>E. coli</i> BL21(DE3) and the elution samples. Target bands (23.6 kDa) can be observed at the position around 20 kDa.</b>
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<p align="center"><b>Fig. 2 SDS-PAGE analysis of protein in lysate of <i>E. coli</i> BL21(DE3) and the elution samples. Target bands (23.6 kDa) can be observed at the position around 20 kDa.</b></p>
  
 
===Reference===
 
===Reference===

Latest revision as of 14:41, 12 October 2022


ttpA-his-myc tag

ttpA-his-myc tag can interact with protein Vp0980 and Myc mouse antibody.

Biology

TTPA

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

In order to get the binding ability of purified Vp0980 to purified ttpA receptor, a Myc-tag was fused to the C-terminal of ttpA-his. This part was constructed on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into E. coli DH5α & E. coli BL21(DE3). The positive transformants were selected by kanamycin, colony PCR and sequencing.

Characterization

1. Agarose Gel Electrophoresis (AGE)

After transforming the plasmid into E. coli BL21(DE3), colony PCR was used to verify that the plasmid was correct. The expected result was obtained (764 bp).

T--XMU-China--089 fig.1.png

Fig. 1 The result of colony PCR.


2.SDS-PAGE

The plasmid 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-myc tag (Fig. 2), the target protein (23.6 kDa) could be observed at the position around 20 kDa on the purified protein lanes (FR).

T--XMU-China--4195089 fig.2 .png

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (23.6 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]