Difference between revisions of "Part:BBa K3128001:Design"

(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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To do a BACTH assay the reporter gene need to :
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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.
 +
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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Have a CAP inducible promoter : PLac
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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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A strong RBS to optimize protein expression
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Those sites were removed after the clonage by side directed mutagenesis.
  
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A easly measurable protein : NanoLuciferase
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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
  
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===Source===
  
 +
PLac-RBS and the termintors are from iGEM plates from Bba_J04450.
  
−
https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png
 
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−
===Source===
 
  
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iGEM plate : Bba_J04450 and Bba_K1680009
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NanoLuc gene is from Promega vector's pNL1.1[Nluc]
  
 
===References===
 
===References===
 +
https://www.ncbi.nlm.nih.gov/nuccore/JQ437370

Revision as of 09:47, 26 September 2019


NanoLuciferase reporter for BACTH assay


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 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.

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