Difference between revisions of "Part:BBa K5520009"
Line 4: Line 4:
 
This part is the rational design of the mutated gene CsnB derived from Marine Bacterium Bacilius SP. BY01 with 6×His tag.
 
This part is the rational design of the mutated gene CsnB derived from Marine Bacterium Bacilius SP. BY01 with 6×His tag.
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5520009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5520009 SequenceAndFeatures</partinfo>
Line 15: Line 12:
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K5520009 parameters</partinfo>
 
<partinfo>BBa_K5520009 parameters</partinfo>
 +
<!-- -->
 +
 +
<!-- Add more about the biology of this part here-->
 +
===Usage and Biology===
 +
==Plasmid construction==
 +
We placed 6×His tag into plasmid PET-28a for protein isolation and purification and tranformed PET-28a-6×His-CsnBK260Y into E. coli BL21 (DE3).
 +
 +
==SDS-PAGE analysis of 6×His-CsnBV186Y==
 +
<p>The purification of the CsnB-K260Y enzyme was accomplished using Ni-NTA affinity chromatography, which facilitated the isolation of the target protein due to the presence of a 6×His tag. The effectiveness of the purification process was confirmed by SDS-PAGE analysis of both the unpurified and purified protein samples. As depicted in the figure below, a specific lane 2 corresponding to the mutant enzyme was observed at approximately 30 kDa in the unpurified sample, consistent with the expected molecular weight. After purification, the mutant enzyme demonstrated a profile reminiscent of the wild-type CsnB, displaying a single band at the same molecular weight as the unpurified enzyme solution.</p>
 +
 +
 +
<body>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/5520/parts/14.png" style = "width:600px"></center>
 +
</div>
 +
</figure>
 +
</body>
 +
</html>
 +
 
<!-- -->
 
<!-- -->

Revision as of 05:36, 30 September 2024

6*His-CsnBK260Y

This part is the rational design of the mutated gene CsnB derived from Marine Bacterium Bacilius SP. BY01 with 6×His tag.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 709
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 718
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 58
    Illegal AgeI site found at 541
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Plasmid construction

We placed 6×His tag into plasmid PET-28a for protein isolation and purification and tranformed PET-28a-6×His-CsnBK260Y into E. coli BL21 (DE3).

SDS-PAGE analysis of 6×His-CsnBV186Y

The purification of the CsnB-K260Y enzyme was accomplished using Ni-NTA affinity chromatography, which facilitated the isolation of the target protein due to the presence of a 6×His tag. The effectiveness of the purification process was confirmed by SDS-PAGE analysis of both the unpurified and purified protein samples. As depicted in the figure below, a specific lane 2 corresponding to the mutant enzyme was observed at approximately 30 kDa in the unpurified sample, consistent with the expected molecular weight. After purification, the mutant enzyme demonstrated a profile reminiscent of the wild-type CsnB, displaying a single band at the same molecular weight as the unpurified enzyme solution.


<body> <figure>

<img src = "14.png" style = "width:600px">

</figure> </body> </html>