Part:BBa_K1022118
pBAD: Ulp: TT: pT7: RBS: His6- SUMO: Magainin
This biobrick codes for the promoter pBAD and Ulp protease followed by pT7 promoter and his6-SUMO tag on the Magainin peptide.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 18
Illegal NheI site found at 938
Illegal NotI site found at 714 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1062
Illegal BamHI site found at 683
Illegal XhoI site found at 692
Illegal XhoI site found at 723 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1247
- 1000COMPATIBLE WITH RFC[1000]
Characterization
For more info, visit [http://2013.igem.org/Team:TU-Delft/Peptides#cleavage TU Delft iGEM13 Wiki]
Cleavage of SUMO from Peptide by Ulp-1 protease
Introduction:
The SUMO-peptide helps in increasing the soluble fraction of peptide but peptides are not biologically active in a fusion, they have to be cleaved from the fusion to get a active peptide fraction. This was achieved by in vivo production on SUMO specific Ulp-1 proteases.
The biobrick BBa_K1022118 was constructed in such a way that the SUMO-peptide production was driven by the strong T7 phage promoter and the Ulp-1 production was driven by arabinose inducible promoter pBad. The plasmid was transformed into an BL21(DE3) pLysS strain. This construct was designed to check whether in vivo cleavage is possible. The main idea of the experiment is to first produce large amount of soluble fraction of SUMO-peptides and the produce the Ulp protease to cleave the sufficiently produced fusion peptides.
The protocol can be seen [http://2013.igem.org/Team:TU-Delft/Protocol_11#protocol_11 here].
Result:
Discussion:
Peptide production
The bio brick BBa_K1022103 is used to produce the SUMO tagged peptide Magainin.
For more info, visit [http://2013.igem.org/Team:TU-Delft/Peptides#cleavage SUMO Cleaved by Ulp 1] on TU Delft iGEM13 Wiki
n/a | pBAD: Ulp: TT: pT7: RBS: His6- SUMO: Magainin |