Difference between revisions of "Part:BBa K2933196"

 
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<partinfo>BBa_K2933196 parameters</partinfo>
 
<partinfo>BBa_K2933196 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of VIM-66 and T7 terminator.It encodes a protein which is VIM-66 fused with His and Sumo tag. The fusion protein is about 40.3 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of VIM-66 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
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===Molecular cloning===
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We insert VIM-66 gene into the standard vector then transfer it into E.coli.
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  [[File:VIM-66-PCR.jpeg|600px|center|]] 
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<p style="text-align: center;">
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'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification

Revision as of 13:22, 21 September 2019


T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+VIM-66+T7 terminator

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+VIM-66)and the biological module can be built into E.coil for protein expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 390
    Illegal NheI site found at 167
    Illegal NheI site found at 1298
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 390
    Illegal BglII site found at 279
    Illegal BglII site found at 1227
    Illegal BamHI site found at 478
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of VIM-66 and T7 terminator.It encodes a protein which is VIM-66 fused with His and Sumo tag. The fusion protein is about 40.3 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of VIM-66 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

We insert VIM-66 gene into the standard vector then transfer it into E.coli.

VIM-66-PCR.jpeg

Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification