Difference between revisions of "Part:BBa K5392020"

 
 
(One intermediate revision by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K5392020 short</partinfo>
 
<partinfo>BBa_K5392020 short</partinfo>
  
−
ZaTdT-R335M is a mutant type of ZaTdT. We hope it can catalyze modified nucleotides. Protein expression will be performed in Escherichia coli BL21.
+
==Description==
 +
To obtain the desired saturation mutagenesis, oligonucleotide primers were designed with degenerate codons. In addition, each single-site saturation mutant was generated according to the PCR-based QuickChange method. The PCR was performed according to the operation manual. The PCR product was digested with DpnI restriction enzyme and transformed into E.coli DH5-alpha competent cells.We will extract the plasmid and sequence it to make sure the mutation was successful.
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a1{width:324px;height:317px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a1">
 +
<img src="https://static.igem.wiki/teams/5392/pet28a-zatdt-r335m-page.png" width="324" height="315"/>
 +
</div></html>
 +
 
 +
==Experiment==
 +
===<strong>SDS-PAGE of ZaTdT-R335M</strong>===
 +
We transfected the Sequencing is correct ZaTdT-R335M plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a2{width:370px;height:208px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a2">
 +
<img src="https://static.igem.wiki/teams/5392/zatdt-r335m.png" width="366" height=204""/>
 +
</div></html>
 +
 
 +
===<strong> Catalytic activity assay of ZaTdT-R335M</strong>===
 +
We transfected the Sequencing is correct ZaTdT-R335M plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a3{width:280px;height:101px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a3">
 +
<img src="https://static.igem.wiki/teams/5392/zatdt-r335m-denaturing-page.png" width="277" height=100""/>
 +
</div></html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:25, 30 September 2024


Vector-ZaTdT-R335M-KanR

Description

To obtain the desired saturation mutagenesis, oligonucleotide primers were designed with degenerate codons. In addition, each single-site saturation mutant was generated according to the PCR-based QuickChange method. The PCR was performed according to the operation manual. The PCR product was digested with DpnI restriction enzyme and transformed into E.coli DH5-alpha competent cells.We will extract the plasmid and sequence it to make sure the mutation was successful.

Experiment

SDS-PAGE of ZaTdT-R335M

We transfected the Sequencing is correct ZaTdT-R335M plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity

Catalytic activity assay of ZaTdT-R335M

We transfected the Sequencing is correct ZaTdT-R335M plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity

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]