Difference between revisions of "Part:BBa K5392017"

 
 
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ZaTdT-R335W is a mutant type of ZaTdT. We hope it can catalyze modified nucleotides. Protein expression will be performed in Escherichia coli BL21.
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==Description==
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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.
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<img src="https://static.igem.wiki/teams/5392/pet28a-zatdt-r335w-page.png" width="324" height="315"/>
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==Experiment==
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===<strong>SDS-PAGE of ZaTdT-R335W</strong>===
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We transfected the Sequencing is correct ZaTdT-R335W 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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<img src="https://static.igem.wiki/teams/5392/zatdt-r335w.png" width="366" height=204""/>
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</div></html>
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===<strong> Catalytic activity assay of ZaTdT-R335W</strong>===
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We transfected the Sequencing is correct ZaTdT-R335W 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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<img src="https://static.igem.wiki/teams/5392/zatdt-r335w-denaturing-page.png" width="277" height=96""/>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:13, 30 September 2024


Vector-ZaTdT-R335W-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-R335W

We transfected the Sequencing is correct ZaTdT-R335W 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-R335W

We transfected the Sequencing is correct ZaTdT-R335W 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]