Part:BBa_K1022117

pBAD: Ulp: TT: pT7: RBS: His6- SUMO: Signiferin

This biobrick codes for the promoter pBAD and Ulp protease with Double terminator followed by promoter pT7 and His 6 SUMO tagged Signiferin peptide.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 18
  Illegal NheI site found at 938
  Illegal NotI site found at 714
  
• 21
  INCOMPATIBLE 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1247
  
• 1000
  COMPATIBLE 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_K1022117 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_K1022102 is used to produce the SUMO tagged peptide Signiferin.