Difference between revisions of "Part:BBa K3128001:Design"
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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 | 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 | ||
| − | BglII and BamHI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] | + | BglII and BamHI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.<br> |
===The plasmid=== | ===The plasmid=== | ||
Latest revision as of 20:21, 17 October 2019
NanoLuciferase reporter for BACTH assay
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
BglII and BamHI restrictions sites were added on the biobrick to to remove the RFP gene and replace it by the NanoLuciferase gene, at 3' and 5' ends respectively.
Primers to add BglII site :
5'-ATGGTCTTCACCTTAGAAG-3'
5'-CTAGTATTTCTCCTCTTTCTC-3'
Primers to add BamHI site :
5'-TAACGCTGATAGTGCTAG-3'
5'-TTAAGCCAAAATACGCTC-3'
BglII and BamHI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.
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]