Difference between revisions of "Part:BBa K1022103"
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'''For more info, visit [http://2013.igem.org/Team:TU-Delft/Peptides#SUMO  TU Delft iGEM13 Wiki]'''
 
'''For more info, visit [http://2013.igem.org/Team:TU-Delft/Peptides#SUMO  TU Delft iGEM13 Wiki]'''
  
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'''Peptide production : '''
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==Peptide production==
  
 
In the project by iGEM 13 Team TU Delft, a SUMO-peptide fusion was opted as a suitable expression system as they make the fusion proteins more soluble. The peptide by itself is not soluble in the cytoplasm but making a fusion of peptide with Small Ubiquitin like Modifiers (SUMO) will increase the solubility of the peptide, thus increasing the cytoplasmic fraction of the peptide.
 
In the project by iGEM 13 Team TU Delft, a SUMO-peptide fusion was opted as a suitable expression system as they make the fusion proteins more soluble. The peptide by itself is not soluble in the cytoplasm but making a fusion of peptide with Small Ubiquitin like Modifiers (SUMO) will increase the solubility of the peptide, thus increasing the cytoplasmic fraction of the peptide.
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The protocol can be seen  [http://2013.igem.org/Team:TU-Delft/Protocol_10#protocol_10 here].  
 
The protocol can be seen  [http://2013.igem.org/Team:TU-Delft/Protocol_10#protocol_10 here].  
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'''Result'''
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'''MIC determination:'''
 
'''MIC determination:'''

Revision as of 13:15, 3 October 2013

pT7: RBS: His6- SUMO: Magainin II

This part codes for the peptide 'Magainin II' tagged with 'His-6-SUMO' molecule. Its production is triggered by IPTG (through pT7 promoter).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 46
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 170
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 355
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

For more info, visit [http://2013.igem.org/Team:TU-Delft/Peptides#SUMO TU Delft iGEM13 Wiki]

Peptide production

In the project by iGEM 13 Team TU Delft, a SUMO-peptide fusion was opted as a suitable expression system as they make the fusion proteins more soluble. The peptide by itself is not soluble in the cytoplasm but making a fusion of peptide with Small Ubiquitin like Modifiers (SUMO) will increase the solubility of the peptide, thus increasing the cytoplasmic fraction of the peptide.

A gene was constructed in such a way that the SUMO-peptide production was driven by the strong T7 phage promoter. This gene containing plasmid was harboured in a BL21(DE3) strain that has lac promoter driven T7 polymerase. Upon induction by IPTG the SUMO peptide fusion is produced as a soluble protein fraction.

The protocol can be seen [http://2013.igem.org/Team:TU-Delft/Protocol_10#protocol_10 here].

Result


MIC determination:

The MIC of peptide on S. delphini, B. subtilis and E. coli was also done. This characterization can be seen in construct BBa_K1022112.

Also visit the [http://2013.igem.org/Team:TU-Delft/PeptideCharacterization Peptide characterization] page on TU Delft iGEM13 Wiki to know more about the MIC experiments.