Difference between revisions of "Part:BBa K3128001:Design"
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===Design Notes=== | ===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. | + | 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.<br> |
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'. | 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 | 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 were removed after the clonage by side directed mutagenesis. | + | Those sites were removed after the clonage by side directed mutagenesis.<br> |
| − | + | Primers to add BglII site :<br> | |
| + | 5'-ATGGTCTTCACCTTAGAAG-3'<br> | ||
| + | 5'-CTAGTATTTCTCCTCTTTCTC-3'<br> | ||
| + | <br> | ||
| + | Primers to ass BamHI site :<br> | ||
| + | 5'-TAACGCTGATAGTGCTAG-3'<br> | ||
| + | 5'-TTAAGCCAAAATACGCTC-3'<br> | ||
| + | <br> | ||
===The plasmid=== | ===The plasmid=== | ||
Revision as of 13:23, 12 October 2019
NanoLuciferase reporter for BACTH assay
Assembly Compatibility:
- 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.
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.
Primers to add BglII site :
5'-ATGGTCTTCACCTTAGAAG-3'
5'-CTAGTATTTCTCCTCTTTCTC-3'
Primers to ass BamHI site :
5'-TAACGCTGATAGTGCTAG-3'
5'-TTAAGCCAAAATACGCTC-3'
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]