Difference between revisions of "Part:BBa K5520008"

 
 
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<partinfo>BBa_K5520008 short</partinfo>
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This part is composed of the rational design of the mutated gene CsnB derived from Marine Bacterium Bacilius SP. BY01 with 6×His tag.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5520008 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K5520008 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-CsnBV186Y into E. coli BL21 (DE3).
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<center><img src = "https://static.igem.wiki/teams/5520/parts/12.png" style = "width:300px"></center>
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<h2>SDS-PAGE analysis of 6×His-CsnBV186Y</h2>
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<p>The purification of the CsnB-V186Y 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 5 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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<center><img src = "https://static.igem.wiki/teams/5520/parts/13.png" style = "width:400px"></center>
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Latest revision as of 05:40, 30 September 2024


6His-CsnBV186Y

This part is composed of 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-CsnBV186Y into E. coli BL21 (DE3).

SDS-PAGE analysis of 6×His-CsnBV186Y

The purification of the CsnB-V186Y 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 5 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.