Difference between revisions of "Part:BBa K5392002"

 
 
(3 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K5392002 short</partinfo>
 
<partinfo>BBa_K5392002 short</partinfo>
  
−
ZaTdT-R335W is a mutant type of ZaTdT. We hope it can catalyze modified nucleotides. Protein expression will be performed in Escherichia coli BL21.
+
==Description==
 +
 
 +
To make the active center more compatible with 3’-O-(2-nitrobenzyl)-modified nucleotides, we attempted to reshape the catalytic cavity using mutagenesis on the residues within 6 Å around 3’-O-(2-nitrobenzyl) and the base moiety. We predicted the 3D structure of ZaTdT by homology modelling and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP. The results of molecular docking indicate that residues Arg335 and Lys337 are closely related to the catalytic activity of ZaTdT.
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a1{width:590px;height:235px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a1">
 +
<img src="https://static.igem.wiki/teams/5392/structure-of-zatdt-and-molecular-docking.png" width="582" height="234"/>
 +
</div></html>
 +
 
 +
Analysis of the molecular dynamics (MD) trajectories showed that R335 and its spatially neighboring residue K337 in ZaTdT contacted the triphosphate group through hydrogen bonds. We anticipate that eliminating the hydrogen bonds binding force between the above residues and the triphosphate group will release the greater freedom of the modified nucleoside within the active pocket, thereby increasing catalytic activity. Consequently, We performed molecular dynamics simulations of the 3’-O-(2-nitrobenzyl)-dATP docked into ZaTdT and ZaTdT-R335W. The predicted results show that above substitution can abolish the hydrogen-bond connection at position 335.
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a2{width:340px;height:250px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a2">
 +
<img src="https://static.igem.wiki/teams/5392/analysis-of-the-molecular-dynamics.png" width="336" height="247"/>
 +
</div></html>
 +
 
 +
==Experiment==
 +
===<strong>Construction of single-site saturation mutant plasmid</strong>===
 +
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.
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a3{width:590px;height:305px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a3">
 +
<img src="https://static.igem.wiki/teams/5392/pet28a-zatdt-r335w.png" width="586" height="300"/>
 +
</div></html>
 +
 
 +
 
 +
===<strong>SDS-PAGE of ZaTdT-R335W</strong>===
 +
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
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a4{width:370px;height:208px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a4">
 +
<img src="https://static.igem.wiki/teams/5392/zatdt-r335w.png" width="366" height=204""/>
 +
</div></html>
 +
 
 +
===<strong> Catalytic activity assay of ZaTdT-R335W</strong>===
 +
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
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a5{width:280px;height:98px;margin:auto;border:3px solid grey}
 +
 
 +
</style><div id="a5">
 +
<img src="https://static.igem.wiki/teams/5392/zatdt-r335w-denaturing-page.png" width="277" height=96""/>
 +
</div></html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:10, 30 September 2024


ZaTdT-mutant type (ZaTdT-R335W)

Description

To make the active center more compatible with 3’-O-(2-nitrobenzyl)-modified nucleotides, we attempted to reshape the catalytic cavity using mutagenesis on the residues within 6 Å around 3’-O-(2-nitrobenzyl) and the base moiety. We predicted the 3D structure of ZaTdT by homology modelling and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP. The results of molecular docking indicate that residues Arg335 and Lys337 are closely related to the catalytic activity of ZaTdT.

Analysis of the molecular dynamics (MD) trajectories showed that R335 and its spatially neighboring residue K337 in ZaTdT contacted the triphosphate group through hydrogen bonds. We anticipate that eliminating the hydrogen bonds binding force between the above residues and the triphosphate group will release the greater freedom of the modified nucleoside within the active pocket, thereby increasing catalytic activity. Consequently, We performed molecular dynamics simulations of the 3’-O-(2-nitrobenzyl)-dATP docked into ZaTdT and ZaTdT-R335W. The predicted results show that above substitution can abolish the hydrogen-bond connection at position 335.

Experiment

Construction of single-site saturation mutant plasmid

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.


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]