Difference between revisions of "Part:BBa K5392016"

 
Line 3: Line 3:
 
<partinfo>BBa_K5392016 short</partinfo>
 
<partinfo>BBa_K5392016 short</partinfo>
  
−
ZaTdT-R335P is a mutant type of ZaTdT. We hope it can catalyze modified nucleotides. Protein expression will be performed in Escherichia coli BL21.
 
  
−
<!-- Add more about the biology of this part here
+
==Description==
−
===Usage and Biology===
+
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-r335p-page.png" width="324" height="315"/>
 +
</div></html>
 +
 
 +
==Experiment==
 +
===<strong>SDS-PAGE of ZaTdT-R335P</strong>===
 +
We transfected the Sequencing is correct ZaTdT-R335P 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-r335p.png" width="366" height=204""/>
 +
</div></html>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K5392016 parameters</partinfo>
 +
<!-- -->
 +
 
  
 
<!-- -->
 
<!-- -->

Revision as of 05:06, 29 September 2024


Vector-ZaTdT-R335P-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-R335P

We transfected the Sequencing is correct ZaTdT-R335P 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]