Difference between revisions of "Part:BBa K5392015"
 
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<partinfo>BBa_K5392015 short</partinfo>
 
<partinfo>BBa_K5392015 short</partinfo>
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Here, we loaded the genes encoding TdT into plasmid vectors. In this way we were able to over-express the ZaTdT in the Escherichia coli BL21.
 
  
 
==Description==
 
==Description==
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We transfected the ZaTdT gene-bonded pET28a plasmid into E.coli BL21(DE3) competent cell. After overnight, Colonies were then picked and performs protein expression. We identified the ZaTdT expression by SDS-PAGE and tested the incorporation of modified nucleotides by purified ZaTdT with polyacrylamide gel electrophoresis.
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We incorporated the sequences of the ZaTdT gene into the pET28a vector. Then the vector plasmid was transfected into E.coli DH5-alpha competent cells for purification and amplification. In this way, plasmids with the ZaTdT gene were obtained.
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<html><style>
 
<html><style>
 
img{margin:auto;}
 
img{margin:auto;}
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#a1{width:350px;height:400px;margin:auto;border:3px solid grey}
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#a1{width:407px;height:262px;margin:auto;border:3px solid grey}
  
 
</style><div id="a1">
 
</style><div id="a1">
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<img src="https://static.igem.wiki/teams/5392/wtzatdt.png" width="350" height="400"/>
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<img src="https://static.igem.wiki/teams/5392/pet28a-zatdt-page-1.png" width="407" height="262"/>
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</div></html>
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==Experiment==
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===<strong>SDS-PAGE of ZaTdT</strong>===
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We transfected the ZaTdT gene-bonded pET28a plasmid into E.coli BL21(DE3) competent cell. After overnight, Colonies were then picked and performs protein expression. We identified the ZaTdT expression by SDS-PAGE and tested the incorporation of modified nucleotides by purified ZaTdT with polyacrylamide gel electrophoresis.
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<html><style>
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img{margin:auto;}
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#a2{width:370px;height:205px;margin:auto;border:3px solid grey}
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</style><div id="a2">
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<img src="https://static.igem.wiki/teams/5392/zatdt.png" width="366" height="201"/>
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</div></html>
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===<strong> Catalytic activity assay of ZaTdT</strong>===
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We transfected the Sequencing is correct ZaTdT plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity
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<html><style>
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img{margin:auto;}
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#a3{width:388px;height:130px;margin:auto;border:3px solid grey}
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</style><div id="a3">
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<img src="https://static.igem.wiki/teams/5392/zatdt-denaturing-page.png" width="388" height=130""/>
 
</div></html>
 
</div></html>

Latest revision as of 15:06, 30 September 2024


Vector-ZaTdT-KanR

Description

We incorporated the sequences of the ZaTdT gene into the pET28a vector. Then the vector plasmid was transfected into E.coli DH5-alpha competent cells for purification and amplification. In this way, plasmids with the ZaTdT gene were obtained.


Experiment

SDS-PAGE of ZaTdT

We transfected the ZaTdT gene-bonded pET28a plasmid into E.coli BL21(DE3) competent cell. After overnight, Colonies were then picked and performs protein expression. We identified the ZaTdT expression by SDS-PAGE and tested the incorporation of modified nucleotides by purified ZaTdT with polyacrylamide gel electrophoresis.

Catalytic activity assay of ZaTdT

We transfected the Sequencing is correct ZaTdT plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity