Difference between revisions of "Part:BBa K3128001:Design"
(→‎Design Notes)
 
(11 intermediate revisions by the same user not shown)
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
To do a BACTH assay the reporter gene need to :
+
[https://parts.igem.org/Part:BBa_J04450 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>
−
Have a CAP inducible promoter : PLac
+
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.<br>
−
A strong RBS to optimize protein expression
+
<br>
−
A easly measurable protein : NanoLuciferase
+
Primers to add BglII site :<br>
 +
5'-ATGGTCTTCACCTTAGAAG-3'<br>
 +
5'-CTAGTATTTCTCCTCTTTCTC-3'<br>
 +
<br>
 +
Primers to add BamHI site :<br>
 +
5'-TAACGCTGATAGTGCTAG-3'<br>
 +
5'-TTAAGCCAAAATACGCTC-3'<br>
 +
<br>
  
 +
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] compatibility.<br>
  
−
https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png
+
===The plasmid===
  
  
 +
https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png
  
 
===Source===
 
===Source===
  
−
iGEM plate : Bba_J04450 and Bba_K1680009
+
PLac-RBS and the termintors are from iGEM plates from Bba_J04450.<br>
 +
NanoLuc gene is from Promega vector's pNL1.1[Nluc]
  
 
===References===
 
===References===
 +
https://www.ncbi.nlm.nih.gov/nuccore/JQ437370

Latest revision as of 20:21, 17 October 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.
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'

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] compatibility.

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