Difference between revisions of "Part:BBa K5520009"
 
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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.
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5520009 SequenceAndFeatures</partinfo>
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K5520009 parameters</partinfo>
 
<partinfo>BBa_K5520009 parameters</partinfo>
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===Usage and Biology===
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==Plasmid construction==
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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).
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==SDS-PAGE analysis of 6×His-CsnBV186Y==
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<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>
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Latest revision as of 05:39, 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.