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

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'.

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

The plasmid

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

@@ Line 9: / Line 9: @@
 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'.
 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
-Those sites are still in the biobrick.
+Those sites were removed after the clonage by side directed mutagenesis.
 ===The plasmid===
 https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png