Difference between revisions of "Part:BBa K5392020"

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]

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 <img src="https://static.igem.wiki/teams/5392/zatdt-r335m.png" width="366" height=204""/>
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+===<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>
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+<img src="https://static.igem.wiki/teams/5392/zatdt-r335m-denaturing-page.png" width="277" height=100""/>
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