Difference between revisions of "Part:BBa K3128004:Design"

 
 
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K3128001 short</partinfo>
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<partinfo>BBa_K3128004 short</partinfo>
  
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<partinfo>BBa_K3128001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3128004 SequenceAndFeatures</partinfo>
  
  
 
===Design Notes===
 
===Design Notes===
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We use the BBA J04450 and remove the RFP gene to put the NanoLuc gene but this part does not contain any restriction site to remove only the RFP. We add 2 restrictions sites on the biobrick to allow us to remove it and put the NanoLuc. BglII in 3' and BamHI in 5'. Those sites are still in the biobrick.
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BBA J04450 was used for its CAP dependant Lactose Promoter (PLac) and it's strong RBS making it able to produce a large amount of protein when activated by IPTG and cAMP-CAP complexes.
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Two restrictions sites were added on the biobrick to to remove the RFP gene and replace it by the NanoLuciferase gene, BglII in 3' and BamHI in 5'.  
  
  
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https://2019.igem.org/wiki/images/thumb/f/f1/T--Grenoble-Alpes--PLac_RFP_%2B_sites.png/796px-T--Grenoble-Alpes--PLac_RFP_%2B_sites.png
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Those sites were removed after the clonage by side directed mutagenesis.
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===The plasmid===
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https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png
  
 
===Source===
 
===Source===
  
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PLac-RBS and the termintors are from iGEM plates
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PLac-RBS and the termintors are from iGEM plates from Bba_J04450.
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The NanoLuc gene is from Promega
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NanoLuc gene is from Promega vector's pNL1.1[Nluc]
  
 
===References===
 
===References===
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https://www.ncbi.nlm.nih.gov/nuccore/JQ437370

Latest revision as of 14:43, 1 October 2019


NanoLuciferase reporter for BACTH assay (with two restriction enzymes around the reporter)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 229
    Illegal BamHI site found at 769
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

BBA J04450 was used for its CAP dependant Lactose Promoter (PLac) and it's strong RBS making it able to produce a large amount of protein when activated by IPTG and cAMP-CAP complexes. Two restrictions sites were added on the biobrick to to remove the RFP gene and replace it by the NanoLuciferase gene, BglII in 3' and BamHI in 5'.


796px-T--Grenoble-Alpes--PLac_RFP_%2B_sites.png

Those sites were removed after the clonage by side directed mutagenesis.


The plasmid

595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png

Source

PLac-RBS and the termintors are from iGEM plates from Bba_J04450.


NanoLuc gene is from Promega vector's pNL1.1[Nluc]

References

https://www.ncbi.nlm.nih.gov/nuccore/JQ437370