Reporter
Plac_NLuc

Part:BBa_K3128001:Design

Designed by: Lucas PINERO   Group: iGEM19_Grenoble-Alpes   (2019-09-15)
Revision as of 08:12, 15 October 2019 by Pinerol (Talk | contribs) (→‎Design Notes)


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'.

Primers to add BglII site :
5'-ATGGTCTTCACCTTAGAAG-3'
5'-CTAGTATTTCTCCTCTTTCTC-3'

Primers to add BamHI site :
5'-TAACGCTGATAGTGCTAG-3'
5'-TTAAGCCAAAATACGCTC-3'

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

BglII and BamHI were removed after the clonage by side directed mutagenesis in order to stay RFC[10] compatible.

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